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1899       A  class  book  of  ele 


RECAP 


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:      ■:.-: 


Practical  Physiology. 


HISTOLOGY, 

CHEMICAL   PHYSIOLOGY, 

EXPERIMENTAL    PHYSIOLOGY. 


MAMMMMM 


de  Burgh   Birch. 


Columbia  ©mbemtp 

tit  tfje  Cttp  of  J8eto  gorfe 

COLLEGE  OF  PHYSICIANS 
AND   SURGEONS 


Reference  Library 

Given  by 


A     CLASS     BOOK 


OK 


(ELEMENTARY) 

Practical    Physiology 

INCLUDING 

HTSTOLOGY,    CHEMICAL    AND    EXPERIMENTAL 

PHYSIOLOGY. 

BY 

DE    BURGH    BIRCH,    M.D.,    CM.,    F.R.S.E., 

PROFESSOR     OK     PHYSIOLOGY     IN     THE     YORKSHIRE     COLLEGE     OK     THE     VICTORIA 

UNIVERSITY;     EXAMINER    IN    VICTORIA    UNIVERSITY;     ADDITIONAL 

EXAMINER     IN    EDINBURGH     UNIVERSITY. 


PHILADELPHIA 

P.    BLAKISTONS   SON   &    CO 

1012    WALNUT    STEEET 
1899 


Printed  in  Great  Britain. 


CHORLET    &    PlCKERSGILL,    THE    ELECTRIC    PRESS,    LEEDS. 


353 


PREFACE. 


This  volume  represents  in  a  more  formal  setting  the  notes 
which  the  author  has  been  in  the  habit  of  issuing  to  students 
attending  the  ordinary  course  of  Practical  Physiology  in  this 
College. 

The  aim  has  been  to  supply  the  student  with  concise 
directions  for  performing  the  work  which  he  has  to  do  with 
his    own   hands   in   class. 

The  choice  of  methods  has  been  necessarily  governed  by 
their  adaptability  to  class  purposes,  and  as  much  as  possible 
also   by  their   simplicity. 

In  order  that  the  student  may  read  beforehand  the  work 
that  is  to  engage  his  attention  at  the  next  meeting  of  the 
class,  the  subject  matter  has  been  divided  into  lessons,  which 
are  indicated   by  marginal   numbers. 

Illustrations  have  been  omitted  from  the  section  on  Histology, 
as  it  is  essential  that  the  student  should  s}^stematically  practice 
drawing  from  the  preparation  itself  uninfluenced  by  the  sug- 
gestions  of    a   drawing   ready  to  his  hand. 

He  is  thus  led  to  form  his  own  opinion  of  what  he  himself 
sees,  and  is  encouraged  to  cultivate  and  to  rely  upon  his  own 
powers   of    interpretation. 

For  the  same  reasons  tracings  have  been  omitted  from  the 
Experimental  Section. 

A  system  of  abbreviated  references  to  the  Appendix  lias 
been  employed  in  those  cases  in  which  sections  of  tissues  are 
given  to  the  class  ready  for  mounting,  by  which  means  the 
methods   of    preparation   can   be   found   in   full. 


IV  PREFACE. 

Much  attention  has  been  devoted  by  the  author  to  the 
simplification  of  the  instrumental  appliances*"-  in  the  Experimental 
Section,  so  that  the  student's  time  may  not  be  needlessly  spent 
in  mastering  details  of  machinery,  which  are  not  the  direct 
objects    of   study  but   only  the   means    to   an   end. 

The  author  is  indebted  to  his  former  demonstrators, 
W.  Gough,  B.Sc.  Lond.,  and  J.  A.  Cairns  Forsyth,  B.Sc. 
(Hon.)  Vict.,  for  repeating  many  of  the  processes  in  the 
Histological  Section,  and  to  his  present  demonstrator,  J.  W. 
Milroy,  M.A.,  M.B.,  CM.,  Edin.,  for  some  suggestions  in  the 
Chemical  Section. 

Existing  works  have  been  freely  consulted,  and  acknowledg- 
ment has  been  made  as  far  as  possible  in  the  context. 

DE   BURGH   BIRCH. 

Yorkshire  College, 

Leeds,  April,  1S'!>9. 


4  Carried  ou1  mainlj  bj  A.  Kershaw,  East  Dorrington  Street,  Leeds. 


CONTENTS. 

Lessons   are   indicated   by   marginal    numbers   in    the   sequence. 


Part  I.— Histological  Section. 

CHAP.    LESSON  PAGE 

Weights  and  measures  ...         ...         ...         ...         ...       3 

Introduction. — The  microscope,  focussing,  care  of. — The 
work  table. — Cleaning  slides  and  covers. — Labelling. — 
Finishing  off  preparations...         ...         ...         ...         ...       5 

Drawing  and  measuring         ...         ...         ...         ...         ...       8 

I.  1.  Introductory  Exercises. — Examination  of  the  object. — 
Application  of  a  cover-glass. — Irrigation. — Various 
fibres. — Starch  ...         ...         ...         ...         ...         ...     11 

2.  Brownian  movement. — Bacteria. — Milk. — Flotation. — 
Staining  on  the  slide. — Mounting  in  balsam. — Mount- 
ing paraffin  sections  ...         ...         ...         ...         ...     12 

II.  3.  Exercises  in  preparing  tissues. — Fixing  and  Hardening. — 
Decalcifying. — Staining  in  bulk. — Embedding. — Cutting 
sections  ...         ...         .'..         ...         ...         ...         ...     16 

Synopsis  of  treatment  in  embedding,  staining,  and 
mounting         ...         ...         ...         ...         ...         ...         ...     27 

III.      4.     Simple   tissues. — Epithelium,    endothelium. — Stratified. — 

Mitosis  ...         28 

5.  Columnar. — Intestinal. — Ciliated. — Ciliary  motion         ...     30 

6.  Connective  tissue. — Areolar. — Tendon. — Action  of  reagents    33 

7.  Tendon,  of  rat's  tail,  of  large  mammal. — Yellow  fibrous. 

— Retiform     ...         ...         ...         ...         ...         ...         ...     34 

8.  Fatty   tissue.  —  Cartilage.  —  Hyaline.  —  White    fibro.  — 

Elastic. — Arytenoid 36 

AA 


VI  CONTENTS. 

CHAP.  LESSON*  PAGE 

39 


IV.        9.  Bone,  adult,  dried. — Softened. — Growing,  head  of. 

10.  Growing,  shaft. — Ossification  of  head. — Marrow    . 

V.  Tooth,  adult.  — Growing  ... 

VI.       11.     Muscle. — Non-striped. — Cardiac  

12.  Striped,  crab's. — Isolated  fibres. — Spindles  ... 


42 

44 
47 

48 


VII.      1 3.     Nerve.  —  Medullated.  —  Osmic.  —  Silvered.  —  Nerve 

trunk. — Non-medullated         ...         ...         ...         ...     51 

14.  Nerve   cells 54 

VIII.  Circulatory  system. — Artery  and  vein.— Distended. — 

Aorta. — Sinus  of  Valsalva. — Ventricle  of  Sheep...     55 

15.  Blood.  —  Amphibian     and     human. — Red     cells. — 

Reagents — Leucocytes...         ...         ...         ...         ...     57 

16.  Fixed     blood.  —  Amoeboid     leucocytes.  —  Fibrin. — 

Haemoglobin,  haemin  crystals  ...         ...         ...     60 

17.  The  circulation  in  the  frog        ...         ...         ...         ...     62 

IX.  Resjriratory    system. — Trachea. — Bronchus. — Lung, 

silvered   ...         ...         ...         ...         ...         ...         ...     65 

18.  Foetal. — Injected 67 

X.  Alimentary  canal. — Tongue. — Papillee. — Soft  palate. 

— Oesophagus     ...         ...         ...         ...         ...         ...     68 

19.  Stomach,     cardio-oesophageal. — Cardiac. — Pyloric. — 

Pyloro-duodenal.  —  Injected.  —  Small     intestine.  — 
Fat  absorption. — Auerbach's  plexus  ...         ...     70 

20.  Peyer's  patch. — Injected. — Large  intestine. — Vermi- 

for  Appendix      ..         ...         ...         ...         ...         ...     74 

XI.  Glands,  parotid. — Sub-maxillary. — Injected...         ...     76 

21.  Pancreas. — Liver,    cells. — Capsule. — Portal    tract. — 

Bile  ducts. — Gall  bladder. — Glycogen        ...         ...     77 


CONTENTS.  Vll 

CHAP.  LESSON  PAGE 

XII.  22.  Kidney,  general. — Papilla. — Boundary  zone.— In- 
jected.— Isolated  tubules. — Embryonic. — Ureter, 
— Bladder 


XIII.  23.     Skin,— Scalp.— Nail         

XIV.  Blood  glands,  Lymph. — Injected. — Tonsil 

24.  Hsemolymph.  — Spleen.  —  Thyroid. — Thymus 

thyroid    ... 

25.  Pituitary  body.  — Suprarenal 


— Para 


80 
84 

86 

87 
89 


XV.  Nervous  system. — Cervical  ganglion. — Dorsal  root. — 

Vagus.  — Spinal  cord.  — Thoracic       ...         ...         ...     91 

26.  Fresh  cord. — Injected. — Conus. — Lumbar. — Cervical. 

— Bulb     93 

27.  Cerebellum. — Pons. — Mid-brain. — Cerebral  cortex    ...     95 

XVI.  28.  Organs  of  generation. — Testis. — Spermatogenesis. — 
Vas  deferens. — Prostate. — Ovary. — Fallopian  tube. 
— Uterus. — Vagina       ...         ...         ...         ...         ...     98 

29.  Mammary  gland 100 

XVII.  Sense     organs,    olfactory.  —  Eye.  —  Corneo- sclerotic 

region. — Cornea. — Lens  ...         ...          ...         ...   101 

30.  Optic     papilla.  —  Retina.  —  Ear,     cochlea.  —  Semi- 

circular canals  ...         ...         ...         ...          ...         •••   103 

Appendix  to  the  Histological   Section. — Preserving, 
fixing,  and  hardening  fluids...         ...         ...         ...   107 

Staining      ...         ...         ...         ...         ...         ...         ...   110 

Injection  with  coloured  gelatin  masses  ...         ...   116 


Part  II.— Chemical  Section. 

XVIII.         1.     Carbohydrates. — Glucoses. — Saccharoses,  cane   sugar. 

— Maltose. — Lactose    ...         ...         ...         ...         ...   121 

2.     Amyloses.  —  Starch.  -  -  Transformations.  —  Precipita- 
tions.— Dextrin...         ...         ...         ...         ...         ...   1*24 


nil 


CONTENTS. 


CHAP.  LESSON 

3.     Glycogen     . . . 


XIX.  Fats. — Saponification. — Emulsification 

XX.       4.     Proteids,  albumins  and  globulins. — Reactions 
5.     Coagulation  temperature. — Albuminates 

Tables    for    the    rough   recognition  of    proteids  and 


PAGE 

.  126 
.  127 
.  129 
.  132 


carbohydrates    ...         ...         ...         ...         ...         ...   135 

XXI.       6.  Some  food  substances.     Milk     ...         ...         ...         ...  136 

7.     Flesh.— Wheat  flour       138 

XXII.       8.  Digestion. — Gastric           ...         ...         ...         ...         ...   140 

9.  Free  hydrochloric  and  lactic  acids. — Pancreatic     ...   142 

XXIII.  10.     Blood.—  Serum      ...         "...  144 

11.  Plasma,  coagulation. — Sugar  in  blood  ...         ...   146 

12.  Hsemacytometer. — Hsemoglobinometer  ...         ...   148 

13.  Spectra  of  blood  ...         ...  ...         ...         ...         ...  152 

XXIV.  14.     Bile 155 

XXV.     15.  Healthy  urine. — Inorganic  constituents           ...         ...  157 

16.  Volumetric  estimation  of  phosphoric  acid    ...         ...   160 

17.  Organic  constituents. — Urea. — Quantitative  estimation  161 

18.  Kjeldahl's  process  164 

1 9.  Creatinin.  —  Uric    acid.  —  Urates.  —  Quadriurates.  — 

Oxalates 167 


XXVI.     20     Abnormal. 
Mucin 


Albumosuria.  —  Hemoglobinuria.  — 
170 


21.     Glycosuria. — Quantitative. — Fermentation 
Urinary  test  table 
List  of  reagents    ... 


..  172 

..  177 
..  178 


CONTENTS. 


IX 


CHAP. 

XXVII. 


Part  III.— Experimental  Section. 

LESSON  PAGE 

1.     Electrical  considerations. — Galvanic  cell. — Amalga- 
mation. — Daniell.  — Leclanche  cell.  — Leads.  — Keys 
— Commutator.  — Hand  electrodes    ... 


XXVIII. 
XXIX. 


2.  Inductorium. — Extra  current    ... 

3.  Dissection  of  the  frog. — Pithing. — Dissection 
Experiments. — Galvani. — Rheoscopic  limb    ... 


183 
188 
195 
198 


XXX. 
XXXI. 


XXXII. 


XXXIII. 
XXXIV. 


4.  Transmission  of  nervous   impulses.  —  Excitation  of 

muscle  and  nerves       ...         ...         ...         ...         ...  199 

5.  Changes  in  the  excitability  of   a  nerve.  —  Unipolar 

stimulation         ...         ...         ...         ...         ...         ...  202 

6.  Arrangements  for  recording      ...         ...         ...  ...  204 

7.  Extensibility  and  elasticity  of  muscle  ...         ...  212 

8.  Single  muscular  contraction  and  latent  period       ...  213 

9.  Tetanus       216 

10.     Fatigue 217 

11.  Influence  of  load  on  work        ...         ...         ...         ...  218 

12.  Heating  and  cooling  muscle     221 

13.  Curara  experiment  ...         ...         ...         ...         ...  223 

14.  Effect  of  Veratria           224 

15.  Experiments  on  nerves  ...         ...  ...         ...         ...  226 

16.  Rate  of  transmission  of  a  nervous  impulse...  ...  228 

17.  Electrotonus          231 

18.  Pfliiger's  law         233 

19.  Demarcation  and  action  currents        ...         ...  ...  236 

20.  Circulatory  system           ...         ...         ...         ...         ...  240 


X  CONTENTS. 

CHAP.  LESSON  PAGE 

21.  Pulse  tracing. — Blood  pressure. — Flow  in  capillaries  242 

22.  The  frog  heart      245 

23.  Effect  of  Atropine  and  Muscarine. — Heat  and   cold  248 

24.  Stannius'  ligature. — Latent  period. — Cardiac  tetanus  249 

25.  Suprarenal  extract  on  the  circulation  ...         ...  250 

XXXV.     26.  Vision.— Field  of  vision  251 

27.  Ophthalmoscope. — Schemer's  experiment      252 

28.  Listing's  diagrammatic  eye. — Marriott's  experiment. 

— Discriminative  power. — Periodic  stimulation  of 
retina       ...         ...         ...         ...         ...         ...         ...  256 

XXXVI.     29.     Cutaneous  senses 259 

Appendix  to  the  Experimental  Section        262 


LIST   OF   ILLUSTRATIONS. 


FIG. 


5. 

6. 

7. 

8. 

9. 
10. 
11. 
12. 
13. 
14. 
15. 
16. 
17. 
18. 
19. 
20. 
21. 
22. 
23. 
24. 
25. 
26. 
27. 
28. 
29. 
30. 
31. 


Scales  and  measures     . . 
Student's  microscope 

(Leitz)*       

Eye-piece  micrometer  . . 
Microscopical     measure 

ment    

Ventral   dissection   of  { 

frog*    

Injecting  apparatus 

Embedding  bath    

, ,  mould 

Microtome,  horse  shoe  . . 
,,  rocking 

, ,  ether  f  reezin 

Mussel     

Moist  cell 
Hot  stage 
Frog  support  . . . 


Hemacytometer  (Zeiss)  §  148 

Spectroscope 152 

Ureameter      163 

Kjeldahl  apparatus       . . .   165 

Glass  seeker    181 

Galvanic  cell 183 

Electrical  keys       186 

Ways  of  using  keys  ...  186 
Pohl's  commutator        ...   187 

Hand  electrodes    187 

Induced  currents 188 

Inductorium 189 

Break  extra  current      ...   191 

Primary  circuit      192 

Dorsal  dissection,  frog*  194 
Muscle-nerve  preparation  197 
Electric  forceps     198 


PAGE 

3 


16 
17 
21 
22 
23 
24 
25 
31 
32 
33 
63 


FIG. 

32. 

33. 
34. 
35. 
36. 
37. 
38. 
39. 
40. 

41. 

42. 
43. 
44. 
45. 
46. 
.47. 
48. 
49. 
50. 
51. 
52. 
53. 
54. 
55. 
56. 
57. 
58. 
59. 
60. 
61. 

62. 


PAGE 

Paradoxical  contraction  200 
Work  table,  experimental  204 
Recording  cylinder       . . .  205 

arm       206 

Smoking  chamber 207 

Time  distribution  board  208 
Diagram  of  Fig.  37        ...  208 

Varnishing  table* 210 

Afterload,  isometric  con- 
traction        220 

Heating      and      cooling 

muscle 221 

Curara  experiment        . . .  223 

Double  electrodes 226 

Pendulum  myograph    . . .  229 

Electrotonus 231 

Pnuger's  law 234 

Galvanometer         238 

Sphygmometer  (Hicks)  §   243 

244 
246 
251 
253 
255 
256 
259 
260 
263 
263 
264 
264 


Glass  staff      

Frog  heart  recorder 

Perimeter        

Ophthalmoscope    

Schemer's  experiment  . . . 

Diagrammatic  eye 

iEsthesiometer       

Aly's  sesthesiometer 

Compensation  method  . . . 

Wheatstone's  bridge     ... 

Paul's  bridge  box  § 

Kohlrausche's  method . . . 

Resistance  galvanometer 
and  cell       

Power  distribution 


265 
266 


The  Author  is  responsible  for  the  actual  drawings,  excepting  those  marked  (*)  or 
which  bear  their  origin  in  brackets  (§). 


ERRATA. 

Pg.  13,  last  line,  for  "translucent"  read  "transparent."  Pg.  32,  5th  from  bottom, 
for  " cilary "  read  "ciliary."  Pg.  97,  2nd  line,  for  "which"  read  "and;"  10th  line,  the 
body  "of  the  cell."  Pg.  98,  last  line  but  one,  for  "acina"  read  "acini."  Pg.  100,  line  10, 
for  "spith"  read  "cells."  Pg.  163,  line  4,  after  "place  it  in  B"  add  "nearly  fill  C  with 
water." 


PART   I. 
HISTOLOGICAL   SECTION. 


British  and   Decimal  Weights  and   Measures,  and 
their   conversion  into  each  other. 


CO 

•  r-4 

O 
P-, 

V 

'o 
> 

r. 

<v 
o 

a 

d 
o 


d 

Eh 


o 

d 

d 

o 

•  i— ( 

d 


to 

CO 


l."[»T 


[••I 


t/j 


o 


ifj 


o 

o 

-+ 

(3D 
O 

w 


3 


o 

i — i 
® 


o 

a, 


73 

d 

o 

Oh 

ffq 


3d 

o 
o 
o 


c> 


O 

o 
o 


c3 

Si 


d       >o 


o 

d 
o 


d 


d 
O 
Pu 


a 
(=! 

d 

o 

o 

CM 

® 

03 

d 


d 
a 

o 

o 
o 


LU^i 


Bach  Student  is  required  to  provide  himself  -with  the 

following1  :— 

1.  A  micposeope,  with  a  high  and  low  power,  a  medium  eye-piece 
and  an  eye-piece  micrometer  ruled  in  squares. 

2.  Slides,  1  in.  by  3  in.,  H  gross,  of  white  glass  not  thicker  than 
1.5  mm  (^in.). 

3.  Cover-glasses.  No.  2  (0-17 mm,  about  r-U  in.)  \  oz.  Circular, 
I  in.  across.  This  shape  is  convenient  if  the  mounting  fluid  necessitates 
ringing  ;  the  square  shape,  which  is  a  little  cheaper,  does  for  balsam- 
mounted  objects. 

4.  Square  labels  for  specimens. 

0.  Needles  mounted  in  handles.  No.  6  "Bet weens"  mounted  in 
ctdarwood  handles,  h  in.  of  the  needle  projecting.  The  soft  wood  allows 
of  the  needles  being  removed  and  replaced  when  soiled. 

6.  Two  glass  pods,  drawn  out  thinner  at  one  end  and  bent  to  an 
obtuse  angle.     This  end  for  use  as  a  section  lifter. 

7.  Seissops,  medium  size  and  sharp  pointed. 

5.  Forceps,  two  pairs,  one  fine  pointed  and  one  of  the  ordinary 
dissecting  kind. 

9.  A  razor  with  a  straight  edge  and  a  stiff  back.  Must  not  be 
hollow  ground. 

10.  Pins,  hedgehog  bristles,  fine  linen  thread  and  silk. 

11.  Two  watch  glasses.  12.  Specimen  tubes,  ^  doz. ,  2  in. 
by  §  in.,  corked. 

]  3.     Wide-mouthed  bottles.    Three  3  oz. ,  screw-capped  or  stoppered. 

14.  Drawing  Book  faintly  ruled  in  1  in.  squares. 

15.  A  case  to  hold  specimens. 

10.     Wash  Leather.     A  piece  4  in.  square  to  wipe  lenses. 

17.     Glass  cloth  for  wiping  slides.     18.     Absolute  alcohol  3  oz. 

19.  Gold  size,  \  oz. ,  and  small  camel  hair  brushes  for  ringing 
preparations. 

Reagents  supplied  on  the  work  tables.  The  ordinary  stains, 
mounting  media,  &c,  are  supplied  on  the  tables  in  the  reagent  stands,  and 
Buch  others  as  occasion  may  require. 

Appliances  supplied  on  the  work  tables.  Two  tin  bowls, 
a  small  bunsen  burner,  cut  blotting  paper,  a  tin  hot  stage,  frog  plate,  zinc 
tray,  horse  shoe  microtome  and  glass  plate.  On  a  side  table  ready  for  use 
are  a  warm  chamber  and  appliances  for  embedding  in  paraffin,  a  Cambridge 
rocking  microtome  and  a  Williams'  ether  freezing  microtome  with  foot 
blower. 


INTRODUCTION. 


The  Microscope.  The  compound  microscope  consists  of  an  eye-pie*  • 
or  ocular  A  and  of  an  objective  B  placed  at  opposite  ends  of  the  body  of 
the  instrument.  The  eye-piece  fits  into  the  draw-tube,  which  forms  part 
of  the  body,  and  by  means  of 
which  the  distance  between 
the  eye-piece  and  objective 
can  be  adjusted.  The  body  C 
is  carried  by  the  pillar  D  and 
is  moved  in  the  direction  of  its 
axis  by  means  of  a  friction 
tube  or  (FlG.  1)  a  rack  and 
pinion,  actuated  by  the  milled 
head  F  for  coarse  adjustment 
and  by  the  milled  head  F'  for 
fine  adjustment.  Beneath  is 
the  stage  S,  and  beyond  this 
the  illuminating  appliance, 
which  consists  of  a  double 
mirror  M,  one  side  of  which 
is  plane  and  the  other  con- 
cave. Between  the  mirror  and 
the  stage  is  the  adjustable 
sub-stage  I  which  carries  the 
diaphragm,  for  regulating  the 
quantity  of  light  admitted  to 
the  object,  and  for  which 
a  condenser  may  be  easily 
substituted.  The  latter,  which 
may  be  termed  an  inverted 
objective,  is  employed  to  pro- 
duce a  more  exact  and  powerful 
concentration  of  light  upon 
the  object  than  can  be  ob- 
tained with  the  concave  mirror  alone,  and  is  required  with  powers  of 
less  than  £  in.  or  4  n,m  focal  distance. 

The  student  will  require  for  work  in  the  histological  class  an  instrument 
yielding  magnifications  of  from  50  to  350  or  400  diameters.  He  will 
obtain  this  by  means  of  a  14  in.  Huvsdienian  ocular  and  objectives  of  1  in. 


Fig.  i.    Student's  Microscope. 


G  INTRODUCTION. 

and  ]r  in.  focus,  using  the  6  in.  or  continental  tube  length  (Swift, 
Beck,  &c. ).  Oculars  Nos.  2  and  4  with  objectives  a3  and  D  of  Zeiss. 
Ocular  No.  3  with  objectives  Nos.  2  and  6  of  Leitz. 

The  last-named  maker  supplies  his  No.  II  b  stand  (Fig.  1)  with 
cylinder  diaphragms  carried  in  a  sub-stage  adjusted  by  a  lateral  screw 
together  with  the  ocular  and  objectives  mentioned,  and  a  double 
nose-piece,  for  £6  5s.  An  iris  diaphragm  can  be  substituted  for  the 
cylinder  form  for  10s.  By  the  subsequent  addition  of  a  Homog.  immersion 
objective  and  a  condenser,  this  instrument  will  fulfil  all  the  requirements 
of  the  student  in  pathology.  As  far  as  my  experience  goes  it  is 
simple,   efficient,  and  well  made. 

Method  to  be  followed  in  focussing'  the  microscope.  Always 
see  that  there  is  sufficient  clearance  beneath  the  objective  before 
placing  an  object  upon  the  stage.  Adjust  the  mirror  so  that  the 
field  is  well  and  uniformly  illuminated.  Use  the  plane  mirror  with  the 
low  power  and  the  concave  one  with  the  high  power,  and  keep  the  centre 
of  the  mirror  in  the  optical  axis  of  the  microscope.  Employ  a  small 
aperture  of  the  diaphragm  with  the  high  power.  Place  the  slide  in  position 
on  the  stage  with  the  left  hand,  and  clamp  it  there  with  the  right 
hand  clip.  With  the  low  power  bring  the  object  sharply  into  view 
by  means  of  the  coarse  adjustment.  To  use  the  high  power  immedi- 
ately afterwards,  revolve  the  nose-piece  ;  the  objective  will  come  into 
place  above  but  close  to  its  position  of  focus.  Move  the  slide  to 
and  fro  on  the  stage  with  the  left  hand,  and  use  the  coarse  adjustment 
until  the  moving  object  is  perceptible  ;  let  the  object  rest,  and  complete 
the  operation  with  the  fine  adjustment.  The  high  power  is  usually 
so  adjusted  on  the  nose-piece  that  it  takes  up  a  position  which  only 
necessitates  a  turn  of  the  fine  adjustment  to  bring  the  object  into 
focus. 

Care  of  the  microscope.  Keep  the  objectives  screwed  to  the 
nose-piece. 

To  detect  the  position  of  dirt.  If  defined  specks,  &c,  arc  seen  in 
the  field  they  are  on  the  eye-piece  and  will  move  when  the  latter  is 
turned.  Raise  the  eye-piece,  partially  unscrew  the  upper  (eye-)  lens,  if 
the  specks  move  with  the  lens,  the  latter  must  be  cleaned,  if  they 
remain  stationary  clean  the  lower  (field-)  lens. 

If  objects  focussed  on  the  stage  appear  dim,  the  objective  is  soiled. 
Remove  it  and  examine  its  front  lens  with  an  inverted  eye-piece  held 
close  to  it.  To  clean  lenses  gently  dust  them  with  a  clean  piece  of 
wash    leather    kept  for  the   purpose.     If   this   fails  moisten    with  water, 


WORKING     ARRANGEMENTS.  7 

dry  by  touching  with  blotting  paper,  and  complete  the  cleansing  with 
wash  leather.  If  oils  or  balsam  are  to  be  removed,  dissolve  with  a 
little  alcohol  or  benzene  and  dry. 

Avoid  lubricating  the  coarse  adjustment,  but  rub  it  clean  with  a 
duster.  If  the  line  adjustment  works  stiffly  cleanse  the  screw  of  dust, 
then  remove  old  oil  with  benzene  and  touch  with  a  little  watchmaker's  oil. 

Arrangement  of  the  work  table.  Place  all  that  is  likely  to 
be  required  ready  to  your  hand.  The  microscope  should  stand  at 
a  convenient  distance  from  the  edge  of  the  table  and  directly  in 
front  of  the  observer.  Its  body  should  be  inclined  whenever  the 
nature  of  the  work  will  allow,  so  as  to  avoid  stooping  the  head 
more  than  is  necessary.  Keep  the  drawing  book  to  the  right  of  the 
microscope.  The  case  for  specimens,  and  a  tray  holding  slides,  covers, 
labels  &c,  are  placed  together,  at  the  further  edge  of  the  table. 
This   will   leave   the   remainder   clear   as   working   space. 

In  the  histological  laboratory  of  the  Yorkshire  College  the  space 
allotted  to  each  worker  is  2  ft.  6  in.  square,  and  between  every  two 
places  are  a  porcelain  sink,  water  tap  (low  pressure,  to  avoid 
splashing)  and  electric  light  (16  c.p.),  available  within  18  in.  of  the 
microscope,  and  shaded,  so  as  to  prevent  direct  illumination  of  the 
worker's   eyes.      Each   student   has   also   a   pedestal   locker. 

To  clean  slides  and  cover-glasses.  Wash  them  in  soap  and  water 
or  benzene  to  remove  grease  or  balsam.  Dry  with  a  thin  glass  cloth.  Thin 
covers,  if  very  dirty,  are  placed  in  strong  sulphuric  acid,  washed  in  water, 
and  drained  on  blotting  paper  before  wiping  ;  the  latter  is  accomplished 
either  in  the  fold  of  a  thin  towel  between  the  thumb  and  forefinger  of  one 
hand,  by  gently  moving  them  upon  each  other,  or  between  two  flat  pieces 
of  wood  tightly  covered  with  wash  leather.  A  stock  of  slides  and  covers, 
ready   cleaned   and    protected   from   dust,  should   be   kept   in   readiness. 

Labelling'  preparations.  Label  without  delay  and  write  in  ink. 
Use  two  labels,  one  at  each  end  of  the  slide.  One  should  bear  the 
serial  number  and  class  designation  or  special  point  illustrated,  written 
as  large  as  the  space  will  allow  for  ease  of  reference,  and  the  name  of 
the  owner.  The  other  should  give  information  as  to  origin,  method 
of  preparation,  the  nature  of  the  stain,  the  nature  of  the  mounting 
fluid,  if   necessary,  and   the   date. 

Finishing  off.  All  preparations  should  be  laid  flat.  Those  mounted 
in  balsam  until  hardened.  Air  spaces,  which  may  arise  in  the  course 
of  drying,  should  be  filled  up  with  balsam.  Any  very  obtrusive 
balsam  may  be  scraped  off  with  a  knife,  and  the  slide  cleaned  with 
a  rag  moistened  with  benzene.     They  should  not  be  ringed.     Specimens 


INTRODUCTION. 


mounted  in  glycerin,  glycerin  jelly,  or  Farrcmt's  solution,  require 
ringing  to  preserve  them,  a  process  much  simplified  by  taking  care 
that  at  the  time  of  mounting,  only  sufficient  fluid  is  used  to  fill  the 
space  between  the  glasses.  The  edge  of  the  cover  and  the  neighbouring 
surface  of  the  slide  must  be  clean,  so  that  the  cement  may  hold. 
Overflow  of  the  mounting  medium  is  first  removed  with  blotting 
paper  moistened  with  water  and  then  with  alcohol.  The  slide  is 
centered  on  a  turn  table,  which  can  be  borrowed  in  the  laboratory, 
and  a  ring  of  thickened  gold  size  applied  with  a  small  brush  whilst 
the  table  revolves  at  a  moderate  speed.  Make  the  ring  as  narrow 
as  is  consistent  with  complete  sealing  and  due  hold  upon  the  glass. 
A  second  application  of  the  same,  or  of  Zinc  white  cement,  will 
complete   the   ringing. 

DRAWING  AND  MEASURING  AN  OBJECT. 

Draw  every  object  which  you  examine  and  append  marginal 
notes  connecting  the  latter  with  the  parts  noted  by  directing  lines. 

Sketches  are  to  enable  you  to  study  later  on  the  preparations  which 
you  have  made  in  class.  It  does  not  matter  how  slight  the  sketch  is, 
provided  the  principal  features  and  their  position  in  the  preparation  are 

properly  noted.  Draw  on  paper  faintly  ruled 
in  squares,1  avoid  a  cramped  style  and  mere 
mechanical  repetition  of  detail.  When  the 
subject  is  the  section  of  an  organ,  draw  the 
general  outline  as  seen  under  the  low  power. 
Naked  eye  examination  is  of  great  help  when 
the  section  exceeds  the  dimensions  of  the  field. 
Then  give  high  power  views  of  those  portions 
which  the  preparation  is  meant  to  illustrate. 
Light  washes  of  water  colour  are  effective 
additions,  especially  if  they  reproduce  the 
colours    of    the    stains    with    which    the    tissues    are    treated. 


Fig.  2.    Zeiss  Eye-piece 
Cross-line  Micrometer. 


An  eye-piece  micrometer'2  is  a<  great  help  as  it  serves  both  as 
a  guide  in  drawing  and  as  a  scale  for  measurement. 

Measurement  of  an  object. — The  simplest  way  is  to  standardise 
the  eye-piece  cross-line  micrometer  for  each  combination  of  eye-piece  and 

objective,  and  to  measure  with  it.     A  stage  micrometer  is  necessary,  and 

1  Suitable  drawing  books  can  l>e  obtained  from  the  Laboratory  attendant,  W. 
Bacon. 

-  Eyepiece  cross-line  micrometer  of  Zeiss,  divided  r.  mm  into  1  »"",  price  5s.  'lids  is 
dropped  into  the  eye-piece,  and  rests  upon  the  diaphragm,  which  must  he  adjusted  hy 
pushing  it  up  or  down  until  the  lines  of  the  micrometer  are  in  the  focusof  the  eye-glass. 


MKASUUKMKXT     UNDKlt     THE     MICKOSCOI'K. 


1) 


can  be  obtained  in  the  laboratory.  This  ia  an  actual  scale  on  glass 
divided  into  fractions  of  a  millimetre  (TJ5)  or  fractions  of  an  inch 
(Tis  and  TuVio)-  With  the  ocular  micrometer  in  the  eye-piece  focus  the 
divisions  of  the  stage  micrometer. 

Using  the  same  eye-piece 
and  the  saim  length  of  tuf>e, 
determine  for  each  of  your 
objectives  the  number  of 
divisions  of  the  stage  mi- 
crometer which  equal  one 
division  of  the  eye  -  piece 
scale,  and  note  the  results 
as  follows  : — One  division 
of  the  scale  in  Oc.  2  with 
Obj.  3  is  equal  to  say  15 
divisions  of  the  stage  scale 
(i5o  mra)  =  0-15  mm,and  with 
Obj.  6  equals  3£  stage  di- 
visions =  0-0325  mm.  The 
same  may  be  done  with  a 
stage  scale  giving  fractions 
of  an  inch.  The  ocular 
scale  is  now  standardised 
for  those  particular  optical 
combinations.  For  example, 
to  measure  the  diameter  of 
a  red-blood  corpuscle  sub- 
stitute a  dried  film  of 
human  blood  for  the  stage- 
scale  and  using  Obj.  6  find 
how  ■  many  red  corpuscles 
in  a  row  fill  one  division  of 
the  ocular  scale.  Assuming 
that  it  takes  four,  the  dia- 
meter of  one  corpuscle  is 
0-0325 1UJU  divided  by  4  = 
0-008 mm  (Sfi).     The  metric 


|    |N.  =  25mm 


Fig.  3.     Measurement,  using  both  eyes. 
R  right,    and  L   left  eye.      M  Microscope.      SM 
Stage    micrometer.       S    Scale    of    inches.       C  Com- 
parison of  the  two  images  in  the  brain.     It  is  presumed 
that  the  magnification  is  such  as  to  produce  a  retinal 
standard     of    microscopical    image  in  the  left  eye  equalling  that  in  the  right  eye. 
measurement  is  the  micron 
0-001 nira  designated  by  the  Greek  /i. 

In  the  absence  of  an  eye-piece  micrometer,  proceed  as  follows  : — -Look 
through  the  microscope  at  the  stagescale  with  the  left  eye,  at  the  same 


10  INTRODUCTION. 

'time,  with  the  right  eye,  look  at  a  centimetre  scale  held  close  to  the 
stage  and  at  a  distance  of  25 cm  from  the  eye.  The  two  scales  appear 
superposed  upon  each  other.  Count  the  stage  divisions  which  exactly 
correspond  to  one  cm.  of  the  centimetre  scale,  which,  with  the  high 
power  we  will  presume  to  be  3,0*03 mm  now  appear  equal  to  10  mm  and 
are  therefore  magnified  333  times,  and  this  is  the  magnifying  power  of 
the  combination.  To  measure  an  object,  compare  its  magnified  image 
with  the  millimetre  scale  and  compute  from  the  ascertained  ratio. 
Instead  of  the  mm.  scale,  the  points  of  a  pair  of  dividers  may  be  held 
in  the  required  position  and  be  made  to  include  a  number  of  the  stage 
divisions.  The  distance  between  the  divider  points  is  then  read  off  on 
the   cm.   scale,  the  computation   being   the   same   as   before. 


CHAPTER   I. 
INTRODUCTORY    EXERCISES. 

The  following  substances  may  be  accidentally  included  in 
a  preparation  and  present  subjects  upon  which  a  number  of 
operations  important  in  microscopy  can  be  usefully  practised. 

Examination  and  description  of  an  object.  As  a  rule  examine 
first  with  a  low  power  {L)  to  gain  an  insight  into  the  general  appear- 
ance and  arrangement  of  the  object,  and  its  apparent  size  under  this 
magnification.  Then  employ  the  high  power  (II)  to  study  detail.  Do 
not  omit  to  return  to  the  low  power  occasionally,  for  the  purpose  of 
comparing  appearances,  and  of  ascertaining  whether  details  found  under 
the  high  power  are  discernible  under  the  lower  one.  This  procedure  is 
of  great  importance  in  accustoming  the  eye  to  recognise  the  relative 
proportions  and  dimensions  of  structures.  In  describing  a  structure 
notes  should  be  made  under  the  following  headings.  Form,  general 
shape,  outlines,  surfaces — under  this  the  nature  of  any  markings  should 
be  given.  Size,  long  and  short  diameters  and  thickness.  Substance, 
whether  uniform  (homogeneous),  granular,  or  reticular.  Presence  of  a 
nucleus  or  other  contents.  Colour.  Grouping,  tendency  to  cohere. 
Movements,  general  and  internal.       Effects  of  reagents. 

Cotton  fibre.  Stretch  a  few  fibres  across  a  drop  of  water 
on  a  slide  and  apply  a  cover. 

Application  of  a  cover-glass.  Always  cover  the  object  with  a 
cover-glass  before  examining  it  under  the  high  power.  Hold  the  cover 
between  the  thumb  and  forefinger  of  the  left  hand,  place  one  edge  of  it 
in  contact  with  the  mounting  fluid,  and  resting  the  other  on  the  point  of 
a  needle  gradually  lower  the  cover  so  that  air  bubbles  may  be  carried  to 
one  side  clear  of  the  object  and  of  the  cover. 

Examine  with  a  low  power  (L),  a  number  of  fine  filaments  are 
visible,  the  details  of  which  are  better  seen  with  a  high  power  (//). 
Each  fibre  is  a  flattened  tube  often  partially  filled  with  air.  The 
walls  are  smooth,  and  the  fibre  is  frequently  twisted. 


1^  INTRODUCTORY    EXERCISES. 

Application  of  a  reagent  by  irrigation.  A  drop  of  the  reagent 
is  placed  on  the  slide  close  to  the  right  edge  of  the  cover,  at  the 
opposite  edge  a  piece  of  blotting  paper  moistened  at  the  tip  is 
laid  in  contact  with  the  fluid  beneath  the  cover.  Guide  the  reagent 
with  a  glass  rod  into  touch  with  the  mounting  fluid,  a  stream  will  be 
established  through  the  preparation  towards  the  blotting  paper.  Different 
fluids  can  be  successively  brought  to  bear  upon  the  preparation  in  this 
manner.  Use  small  quantities  of  the  reagent  and  avoid  staining  the 
stage  of  the  microscope. 

Irrigate  the  preparation  with  Iodine  solution,  the  fibres  will 
be  stained  of  a  slightly  yellow  colour. 

Irrigate,  in  addition,  with  strong  Sulphuric  Acid,  the  fibres 
will  swell  and  turn  blue  ;  this  is  the  reaction  for  Cellulose. 

Linen  fibre.  (//)  The  fibres  are  solid  cylinders  with  a#  smooth 
surface. 

Woollen  fibre.  (//)  Cylindrical  filaments,  the  surfaces  of 
which  show  transverse  markings  which  indicate  their  structure 
of  imbricated  scales. 

Starch,  granules.  Scrape  the  cut  surface  of  a  potato  lightly 
with  the  edge  of  a  scalpel  and  diffuse  the  scraping  in  water. 
(//)  The  oval1  starch  granules  exhibit  concentric  contour  lines 
around  a  spot  placed  near  one  end.  Irrigate  with  Iodine  and 
note  the  blue  colour  produced,  Iodide  of  Starch. 

Brownian  movement.  Rub  a  piece  of  Gamboge  on  a  slide 
in  a  drop  of  water  until  the  latter  has  a  yellow  tint,  cover. 
(//)  Fine  particles  of  various  sizes  are  found  which  exhibit 
oscillatory  movements.  This  phenomenon  is  commonly  exhibited 
by  inanimate  particles  suspended  in  water. 

Bacteria.  (H)  Examine  a  drop  of  fluid  from  an  aqueous 
infusion  of  meat   or  of  chopped   straw   which   has    had    time   to 

1  ']'<>  ascertain  the  shape  <>f  a  small  object  floating  in  a  fluid,  touch  the  cover 
with  a  needle  and  note  any  change  of  configuration  whilst  the  object  revolves  in 
the  field. 


MILK.— -MOUNTING     BY     FLOTATION.  13 

decompose.  Numbers  of  minute  rod-shaped  bodies  {Bacteria) 
appear  moving  through  the  fluid  with  an  undulating  or  spiral 
motion.  They  are  propelled  in  a  definite  direction  by  a  flagellum 
at  one  or  both  ends  ;  these  flagella  are  only  revealed  by  the 
highest  powers.  The  bacteria  are  the  causes  of  the  putrefactive 
changes  in  the  infusion. 

Milk.  A  thin  film  (//)  exhibits  small  spherical  bodies  (fat 
globules)  in  great  numbers,  floating  in  a  colourless  fluid  (milk 
plasma).  The  globules  are  transparent  and  do  not  adhere  to 
each  other.  Irrigate  with  acetic  acid  and  note  the  change 
produced  in  the  behaviour  of  the  globules  to  each  other. 

Newt's  moult.  Squamous  epithelium.  Mount  a  piece, 
which  has  been  preserved  in  alcohol,  as  follows  : — 

Flotation  on  water.  Drop  the  object  into  a  basin  of  water,  the 
effect  of  alcohol  in  unfolding  and  spreading  out  a  thin  film  of  tissue  is 
instantaneous,  the  tissue  lies  on  the  sitrface  of  the  water.  Pass  a  slide 
two-thirds  under  water  in  a  sloping  position,  guide  the  object  to  its 
centre  with  a  needle,  and  holding  it  there  lightly,  raise  the  slide  from 
the  basin.  Then  turning  the  slide  into  the  vertical  position  so  as  to 
drain  the  water  to  one  end,  dry  the  slide  with  blotting  paper  on  Loth 
sides  and  right  up  to  the  edge  of  the  tissue. 

Stain  on  the  slide  with  hematoxylin, ]  by  adding  a  small  drop 
of  the  stain  to  an  equal  quantity  of  water  on  the  slide  and  guiding  it 
over  the  preparation  with  a  glass  rod.  Cover  and  watch  the  develop- 
ment of  the  staining  under  the  low  power,  and  as  soon  as  the  nuclei 
are  sufficiently  tinged,  bathe  off  the  superfluous  colouring  matter  with 
water,  or  float  the  tissue  off  the  slide  into  the  water  and  back  on  to 
the  slide. 

Mount  in  balsam  which  is  the  next  step,  dehydrate  by  dropping 
absolute  alcohol,  6  or  7  drops  successively,  on  the  preparation,  flowing 
it  slowly  across  it  so  as  to  give  the  alcohol  time  to  abstract  the  water. 
The  next  step  is  to  clear  with  oil  of  cloves  (or  other  essential  oil)  by 
placing  a  drop  of  the  oil  on  the  slide  and  guiding  it  to  the  edge  of  the 
object,  it  will  flow  beneath  it,  will  soak  up  through  the  preparation, 
and  will  render  it  translucent.      This   indicates   that   the   oil    has  taken 

lKleinberg's  solution  see  appendix   20. 


14  INTRODUCTORY    EXERCISES. 

the  place  of  the  alcohol.  Observe  this  taking  place  under  a  low  power. 
Should  there  be  any  trace  of  opacity  left  it  is  due  to  incomplete 
removal  of  water.  Further  treatment  with  alcohol  is  necessary  and 
clearing  must  again  be  performed,  and  if  then  there  are  no  signs  of 
opacity,  permanent  mounting  in  balsam  is  finally  performed  by  removing 
superfluous  essential  oil  with  blotting  paper,  adding  a  drop  of  balsam 
and  covering. 

Examine  with  a  high  power  {H)  and  observe  the  polygonal 
cells  joined  edge  to  edge  into  a  continuous  sheet,  each  with  a 
violet  stained  nucleus.  Two  or  three  layers  of  cells  may  be 
found  superposed  upon  each  other. 

Stain  another  piece  with  Picpoeapmine  by  covering  it  with  a 
pool  of  the  reagent,  give  the  stain  five  minntes  to  penetrate,  remove 
the  excess  with  blotting  paper,  and  apply  Glycerin  or  Fappant, 
just  enough  to  fill  the  interval  between  slide  and  cover.  As  the 
carmine  effect  does  not  develop  for  some  time,  it  is  necessary  to 
leave  some  of  the  stain  in  the  preparation,  which  will  become  more 
differentiated  by  the  selective  activity  of  the  tissues  in  the  course  of 
a  week.  Picrocarmine  is  a  double  stain,  the  carmine  colouring  the 
nuclei  and  connective  tissue  where  such  is  present  pink,  and  other 
parts  yellow. 

Mounting-  sections  of  tissues  cut  in  paraffin  and  which  are 
still    permeated  by  the  paraffin  in  which  they  have  been  embedded. 

As  in  the  sequel  the  majority  of  the  sections  given  out  to 
the  class  will  be  of  this  nature,  the  student  should  note  cor<- 
fully   the   uses  of  the  following  alternative  method*. 

1.  Simple  treatment.  If  the  section  be  flat,  i.e.,  is  not  curled  or 
crumpled  beyond  a  very  slight  extent,  and  its  parts  do  not  tend  to 
fall  asunder  when  the  support  of  the  paraffin  is  removed,  proceed  as 
follows  : — Place  the  section  in  the  centre  of  the  slide,  warm  its  under- 
side over  the  burner  just  sufficiently  to  melt  the  paraffin  and  wash  the 
latter  away  with  five  or  six  successive  drops  of  turpentine  or  toluene, 
giving  each  drop  time  to  act.  In  warming  over  the  burner  care  must 
be  taken  not  to  overheat  or  the  tissue  will  be  spoilt.  The  thickness 
of  the  slide  affects  the  time  which  the  heat  takes  to  reach  the  film  of 
paraffin,  therefore  pass  the  slide  twice  immediately  over  the  flame  and 
wait  a  few  moments  to  see  if  the  paraffin  is  going  to  melt,  testing  the 
temperature  of  the  slide  meanwhile  on  the  back  of  the  hand  for  future 


MOUNTING    SECTIONS     CUT     IN     PARAFFIN.  15 

guidance.  Heat  again  if  necessary  until  the  melting  point  is  reached 
and  apply  the  solvent.  Dry  the  slide  with  blotting  paper,  apply  balsam 
and  cover. 

2.  If  the  section  is  to  be  stained1  remove  the  solvent  with  absolute 
alcohol.  Any  trace  of  paraffin  remaining  in  the  section  will  appear 
white,  spicular  under  the  microscope,  and  must  be  removed.  Then 
having  bathed  the  preparation  with  a  drop  of  water,  to  be  certain  that 
it  will  Met  uniformly,  apply  the  staining  reagent. 

3.  Flattening  on  Oil.  Crumpled  sections  which  do  not  recpiire  fixing 
to  the  slide  are  most  conveniently  treated  as  follows  : — A  pool  of  the 
essential  oil  used  for  clearing  (cedar  oil  when  eosin  is  present)  sufficient 
to  float  the  section  is  placed  upon  the  slide  and  is  slightly  warmed. 
The  section  is  floated  upon  it  and  flattens.  The  superfluous  oil  is 
drained  off  with  blotting  paper,  additional  heat  is  applied  just  sufficient 
to  melt  the  paraffin,  drain,  add  more  of  the  oil  to  dissolve  what  remains 
of  the  paraffin,  cool  and  if  no  paraffin  is  visible  add  balsam  and  cover. 

If  the  sections  are  extremely  thin  and  tend  to  fall  apart  when  the 
paraffin  is  melted  employ  one  of  the  following  methods: — 

4.  Shellac  fixation.  For  stained  sections.  Smear  a  very  thin  film  of 
a  saturated  solution  of  white  shellac  in  creosote  with  your  finger  on  the 
middle  of  a  slide,  place  the  section  in  position  and  press  it  lightly  into 
contact  with  the  fixative  with  a  dry  finger.  Melt  the  paraffin  over  the 
burner,  being  careful  not  to  heat  beyond  the  melting  point  ;  keep  it 
melted  for  a  minute,  drain  off  and  dissolve  away  the  remaining  paraffin 
with  turpentine  or  toluene,  apply  balsam  and  cover. 

5.  Water  fixation.  For  sections  which  are  to  be  stained  upon  the 
■slide  {Gulland.)  If  the  section  is  curled  or  frilled,  place  it  on  warm 
water,  not  above  40°  C  ;  it  will  flatten.  Float  on  to  a  slide  or  cover- 
glass,  and  blot  off  the  water  thoroughly.  Heat  to  the  warmth  of  the 
hand  for  ten  minutes  over  the  bunsen  flame  to  drive  off"  all  moisture, 
then  heat  sufficiently  to  melt  the  paraffin.  The  section  will  adhere  to 
the  slide.-  Use  turpentine  to  dissolve  the  paraffin,  blot  off  the  excess, 
add  balsam,  and  cover.  Unstained  sections  after  being  freed  of  tur- 
pentine or  toluene  by  means  of  alcohol  can  be  stained  in  position  upon 
the  slide. 

1  These  operations  can  be  performed  upon  a  large  number  of  sections  at  a  time 
in  a  watch  glass  or  porcelain  capsule.  Heating  unnecessary  provided  sufficient 
solvents  and  time  are  given. 

-  24  hours  drying  at  the  ordinary  temperature  and  shielded  from  dust  is  more 
certain.  The  slides  must  be  perfectly  clean  and  free  fiom  grease.  It  is  best  to 
clean  them  with  alcohol  before  use. 


3  CHAPTER   II. 

EXERCISES  IN  THE   PREPARATION  OF  TISSUES. 


FIG.  4.       Ventral  dissection  of  a  frog. 
St  Sternum  reflected.  B  Bulbus  arteriosus  with 

aorta-,  to   its    right    the    left   auricle,  below    is 

the  ventricle  surrounded  by  P  the  pericardium. 

La  Larynx.  Lg  Lung.  L  right  lobe  of  the  liver. 

P  is  on  the  left  lobe.    GB  Call  bladder,  ST 

Stomach,   Pa   1'ancreas  with  tin-  common  bile 

and  pancreatic  duct  opening  below  into  SI  the 

-mall  intestine.     Between  Si  and  C  the  cloaca 

the  right  testis  shows   through    the  mesentery. 

Aleve  C  is  the  spleen.     V  the  bilobed  bladder, 

distended     with    air.       Muscles     of     the    thigh:— GL    Gluteus,    VI     Vastus    interims, 

AL   Adductor  longns,    S    Rartorius,   AB    Adductor    brevis,    AM    Adductor    magnus, 

RIM  Rectum   interims   major,  below   it  in  the  leg  the  gastrocnemius. 


INJECTION     WITH     CORROSIVE     SUBLIMATE. 


17 


Injection  of  a  frog'  with  corrosive  sublimate  and  preparation 
of  its  tissues  for  microscopical  examination.  A  frog,  lulled  with  ether,  is 
laid  on  its  back  on  a  frog  plate  in  a  zinc  tray.  Divide  the  skin  in 
the  middle  line  from  the  centre  of  the 
abdomen  to  within  half  an  inch  of 
the  mouth,  raise  the  skin  and  note 
where  there  are  large  cutaneous  vessels, 
make  two  transverse  cuts  so  that  the 
skin  can  be  turned  back  freely,  and 
open  the  body  cavity  near  the  middle 
line,  avoiding  the  central  vein  in  cutting 
through  the  muscles  of  the  abdomen. 
Next  raise  the  sternum  by  its  cartilage 
with  forceps,  free  it  and  divide  the  ribs 
on  each  side,  and  turn  it  upwards,  thus 
gaining  free  access  to  the  heart.  Open 
the  pericardium,  hold  the  heart  so  as 
to  put  the  aorta?  on  the  stretch,  and  with 
fine  forceps  pass  a  thread  under  each 
near  its  origin,  scraping  through  the 
confining  connective  tissue  to  do  so. 
Form  a  loop  on  each  thread  ready  for 
tightening.  Next  raise  the  heart,  snip 
into  the  sinus  with  scissors,  cut  off  the 
apex  of  the  ventricle,  and  let  all  the 
blood  escape.  Wash  away  blood  from 
the  body  cavity  with  normal  saline. 
Tie  the  right  aorta,  and  into  the  left 
introduce  a  fine  glass  cannula  through 
the    opening     in     the     ventricle.      The 


Fig.  5.    Simple  class  appliance  for  fixing  animal  tissues  by  injection. 

1  The  same  principles  will  apply  for  injecting  any  small  mammal,  i.e.,  mouse,  rat, 
small  kitten  or  rabbit,  or  a  separate  organ. 


18  EXERCISES    IN    THE    PREPARATION    OF    TISSUES. 

cannula  filled  with  salt  solution  is  passed  in  as  far  as  it  will  go,  and 
is  tied  in  by  tightening  the  loop,  secure  this  by  a  second  knot.  Fill  the 
cannula  completely  with  salt  solution  by  means  of  a  fine-pointed  pipette 
to  displace  air  bubbles,  and  slip  the  short  rubber  connection  of  the 
glass-pressure  apparatus  on  to  the  cannula.  The  latter  must  also  be 
completely  filled  with  salt  solution  before  connecting.  The  salt  solution 
is  now  allowed  to  flow  into  the  vessel  by  opening  the  clip  on  the 
pressure  tube,  and  the  remaining  blood  is  to  be  washed  out.  As 
soon  as  the  fluid  comes  only  faintly  tinged  with  blood  from  the 
sinus  stop  the  injection  and  substitute  saturated  corrosive  sublimate 
solution  in  normal  saline.  To  do  this  detach  the  pressure  tube 
from  the  cannula,  empty  and,  after  refilling  with  corrosive,  re-attach 
to  the  cannula,  being  careful  not  to  displace  the  latter  during  the 
operation.  The  corrosive  is  now  run  in.  The  tissues  will  turn  white 
when  reached  by  the  reagent,  and  the  injection  will  be  complete. 
If  necessary  increase  the  pressure  by  raising  the  thistle  tube.  A 
column  of  pressure  nearly  two  feet  in  height  can  be  brought,  to  bear, 
but  less  than  this  will  be  required. 

After  20  minutes  open  the  body  cavity  freely,  rinse  the  surface  of 
the  organs  with  water,  and  quickly  remove  the  parts  required  by 
cutting  them  out  with  scissors.  (Metal  instruments  are  blackened  by 
f-orrosive.) 

Remove  the  following  organs  : — {a)  Liver,  Kidneys,  Small  Intestine, 
Stomach,  Vago-sympathetic  Ganglion,  Gastrocnemius  Muscle,  (h)  the 
upper  half  of  the  femur  including  its  head,  and   (o)  both  Eyes. 

After    Hardening- 

Put  (a)  and  (c)  into  a  bottle  with  50 cc-  of  70 t>c-  alcohol  tinted  with 
iodine  to  a  sherry  colour,  the  latter  loses  colour  so  long  as  uncombijicd 
corrosive  is  present  and  must  be  renewed  with  each  change  of  spirit  until 
the  discoloration  ceases.  The  iodine  helps  to  remove  the  corros 
forming  iodide  of  mercury.  The  alcohol  is  changed  every  12  or  24 
hours,  each  24  hour's  change  being  1Qp-c-  stronger  than  the  preceding, 
until  full  strength  methylated  spirit  is  reached.  Label  the  bottle  for 
guidance  as  follows  : — 

Always  employ  this  system  of  labelling  when  carrying  out  a 
process. 


DECALCIFICATION.    -STAINING     IN     BULK. 


19 


ORGANS 

(FROC)     NaCl.  Corr.   Inj. 

May   ID 

70  p*-  Ale.  +  I. 

„      11 

change  to  80          ,, 

,,      12 

90 

„      13 

,,          Spt.  Meth. 

No. 

Name 
— — —— . 

Draw  your  pen  through  each  direction  as  the  change  is  made. 
The  tissues  will  be  ready,  at  the  earliest,  in  four  days  for  embedding 
and  cutting.  Longer  exposure  to  spirit  will  confer  a  beneficial  tough- 
ness  upon    the    tissue. 

These  and  the  remaining  steps  in  the  'preparation  of  tissues  are 
ax  far  as  possible  spread  over  subsequent  meetings  of  the  class. 
In  the  intervals  due  attention  must  be  given  to  the  tissues  which 
are  in  the  course  of  preparation,  i.e.,  changing  of  fluids,  <kc. 
The  reagents  mentioned,  ivith  the  exception  of  absolute  alcohol,  are 
provided  in  the  laboratory. 

Decalcification    of    osseous    tissue. 

Place  (b)  in  bone  softening  fluid  (14a,  see  Appendix).  Examine  in  24 
hours,  [and  as  soon  as  it  becomes  pliant  or  feels  soft  to  a  needle  point 
introduced  into  it,  wash  in  changes  of  water  until  the  colour  is  removed 
and  after-treat  with  alcohol.  Bones  are  usually  best  cut  in  frozen  gum, 
but  this  specimen  being  small  will  cut  well  in  paraffin. 

Staining-    in    bulk. 

Small  pieces  are  next  to  be  stained  in  bulk,  as  follows :  cut 
parallel  sided  slices  or  rectangular  blocks  not  more  than  3 mm  thick 
(BHHHH);  the  other  dimensions  being  governed  by  the  nature  of  the 
organ.  Put  one  set  into  a  tube  with  10 cc  hajmalum  solution  (22)  and 
another  into  a  like  quantity  of  borax  carmine  (17)  in  which  thev 
remain  48  hours  or  more,  according  to  their  density.  The  first  are  then 
thoroughly  washed  in  water  for  12  hours  to  remove  the  superfluous 
stain,  and  are  then  placed  in  70p-c-  alcohol  and  through  increasing 
strengths    to  absolute  alcohol.     They  may  be  ground-stained  with   eosin 


20  EXERCISES    IN    THE    PREPARATION    OF    TISSUES. 

by  tinging  the  last  alcohol  with  the  stain.  Those  treated  with  borax 
carmine  are  placed  in  acid  alcohol  (17)  for  24  hours,  this  makes  the  red 
brighter,  and  thence  are  passed  through  increasing  strengths  of  alcohol 
as  in  the  former  case. 

Section   Cutting-. 

The  razor  must  have  a  keen  straight  edge  and  a  stiff  back, 
and  must  not  be  hollow  ground. 

Hand  cutting?'  Holding  the  piece  of  tissue  in  the  left  hand, 
between  the  thumb  and  forefinger,  and  the  razor  in  the  right,  guide 
the  razor  upon  the  forefinger  of  the  left  and  cut  thin  slices  by 
steady  heel  to  point  strokes  of  the  razor.  Keep  the  blade  of  the 
razor  and  the  surface  of  the  tissue  moistened  with  spirit  or  other 
fluid  in  which  the  tissue  happens  to  be  preserved.  The  piece  of 
tissue   may   be   held    between   pieces   of    hardened    liver   or   elder  pith. 

As  tissues  generally  require  support,  owing  to  the  delicateness  of 
their  structure,  it  is  usual  to  embed  them  in  a  material  which  will 
permeate  them.  In  order  to  make  even  and  thin  sections  a  microtome 
is   employed.      Three  principal  methods   are   in   use   as   follows*: — 

Employ  the  prepared  tissues  of  the  frog  in  illustration  of  the 
following  methods,  thus  : — 

Tissues  (a)  and  (b)  are  to  be  cut  in  paraffin. 
„       (a)  „  „         gum. 

„        (r)  ,,  „  celloidin. 

Embedding-  and  cutting  in  paraffin.1 

Transfer  the  pieces  of  tissue  into  fifteen  times  their  volume  of  absolute 
alcohol  for  24  hours  in  order  to  complete  their  dehydration.  Then 
"clear"  them  by  a  corresponding  immersion  in  toluene.  This  is  for 
general  purposes  the  best  intermediate  solvent.  This  step  is  required 
because  alcohol  will  not  mix  with  paraffin. 

When  cleared  (24  hours)  they  are  ready  for  embedding.  Wipe  oil'  the 
surface  toluene  and  immerse  the  pieces  in  melted  paraffin  (melting  point 
55°  C.)   for  one  hour  to  an  hour  and  a  half  in  the  embedding  bath. 

1  Very  delicate;  tissues  require  to  be  guarded  from  the  collapse  of  cavities  which  is 
;>1>1  to  occur  with  this  process  by  a  more  gradual  transference  to  paraffin,  this  is  accom- 
plished by  transferring  from  alcohol  to  chloroform,  and  when  the  latter  has  quite 
replaced  the  alcohol,  the  tissue  sinks  in  it.  Then  place  in  a  fresh  quantity  of 
the  fluid  and  add  shavings  of  solid  paraffin  until  no  more  dissolves.  Allow  the 
fluid  to  thicken  by  evaporation,  warm  to  the  temperature  of  the  embedding  bath, 
and  transfer  to  pure  melted  paraffin  in  the  latter. 


EMBEDDING      IX      l'.\  ICAKI'IN. 


21 


FIG.  6.  Embedding  Bath  for  the  use  of  students  in  class,  Yoikshire  College. 
Around  a  central  water  cistern  are  six  shallow  closets  with  shelves.  Each  closet 
has  a  glass-panelled  door,  over  which  a  non-conducting  flap  C  hangs,  removed  from 
the  front  closets  for  clearness.  R  gas  from  S  supply  to  regulator  and  thence  to 
B,  a  Fletcher's  safety  burner ;  all  metal  piping.  T  thermometer.  (Made  to  the 
author's  directions  by  Messrs.  Braithwaite  &  Co.,  Swinegate,  Leeds.) 


99 


EXERCISES    IN    THE    PREPARATION    OF    TISSUES. 


Blocks  for  cutting  are  now  made  by  forming  cells  with  a  couple  of 
L-shaped  strips  of  lead  resting  on  a  glass  plate.  Into  these  paraffin  is 
poured  to  the  top,  and  the  piece  of  tissue  is  submerged,  with  warmed 
forceps    or   needles,  into  the  position   required  for  cutting.       When   the 


Fi<;.  7.     Mould  of  L-shaped  strips  of  metal  for  embedding  in  paraffin. 

surface  of  the  paraffin  has  solidified  plunge  the  whole  cell  into  cold 
water  to  complete  the  solidification,  then  strip  the  block  of  its*  cell  and 
it  is  ready  to  attach  to  a  microtome. 

Small  pieces  of  fresh  tissue  of  this  thickness  (■OBI)  may  be  dehydrated, 
penetrated  with  clove  oil,  and  saturated  with  paraffin  by  20  minutes' 
exposure  to  each  reagent,  assisted  by  slight  warmth.  If  ten  times  the 
volume  of  the  tissue  be  used,  and  the  fluids  are  individually  changed  three 
successive  times,  a  block  can  be  made  ready  for  cutting  in  about  an 
hour. 

Horse-shoe  microtome.  Clamp  the  razor  blade  in  position  with 
its  shoulder  clear  of  the  frame.  Raise  the  regulating  screw  until 
the  back  of  the  frame  is  close  to  the  glass;  the  razor  edge  is  now 
fully  elevated  above  the  glass  plate,  from  this  the  height  of  the 
paraffin  block  is  gauged.  Pare  the  latter  to  the  required  thickness,  and 
attach  it  to  the  glass  plate.  The  glass  must  be  free  of  moisture ; 
melt  the  block  to  it  with  a  hot  wire  or  blade  of  an  old  scalpel,  and 
bank  some  paraffin  around  its  foot  to  give  a  broad  base  of  attachment. 
Trim  the  front  and  back  faces  of  the  block  straight  and  parallel  to  each 
other.  Secure  the  glass  plate  to  the  table  with  a  piece  of  moist 
blotting  paper.  The  left  hand  holds  the  carrier,  the  thumb  and 
forefinger  of  the  right  hand  grasp  the  regulating  screw,  the  other 
fingers  lying  on  the  frame  as  an  additional  guide,  the  wrists  rest  upon 
the  table  and  by  a  simultaneous  movement  of  both  hands  the  frame  is 
carried  forward    with    a  quick  and  even   swing   to  which   the  weight  of 


HORSE-SHOE     AND     R0CK1N<J     MICROTOMES. 


T.\ 


the  frame  adds  steadiness.  As  the  frame  is  carried  back  for  the  next 
stroke  the  regulating  screw  is  turned  clockwise  through  a  small 
fraction  of  a  turn  by  the  fingers  which  grasp  it,  this  lowers  the  razor 
edge  and   determines    the   thickness   of    the   next   section.     By   keeping 


Fig.  8.     Method  of    using  the  horse-shoe    microtome. 

the  wrists  firmly  upon  the  table  the  same  part  of  the  razor  edge 
is  used  at  each  successive  cut,  a  matter  of  importance,  as  the 
edge  is  generally  curved,  and  lateral  displacement  would  vary  the 
thickness.  Short  ribbons  of  six  or  seven  sections  adhering  to  each 
other   may  be   cut. 

Imperfect  embedding,  causing  want  of  homogeneity,  and  grittiness  in 
cutting,  is  due  to  imperfect  removal  of  water  and  alcohol  in  the  substitution 
processes,  and  can  only  be  cured  by  retrograding  through  the  successive 
steps  to  alcohol  and  back  again  to  paraffin. 

Blocks   and   sections   can  be  preserved   in  pill   boxes  for  later  use. 

Rocking-  microtome.  (Cambridge  Instruments  Company.)  This 
instrument  cuts  ribbons,  i.e.,  successive  sections  adhere  to  each  other. 
The  block  of  paraffin  containing  the  tissue  which  is  to  be  cut  is 
fastened  to  a  brass  thimble  by  means  of  a  hot  wire,  and  its  front 
and  back  edges  are  pared  straight  and  parallel  to  each  other.  Slip 
the  thimble  A  into  position  on  the  end  of  the  rocking  arm  B. 
From  the  other  end  of  B  a  cord  passes  from  a  clamping  ring  over 
guide  pulleys  to  the  lever  C.     By  the  to   and   fro    movement   of   C,    A 


24 


EXERCISES    IX    THE    PREPARATION    OF    TISSUES. 


is  caused  to  make  up  and  down  strokes.  The  latter  movement  cuts 
the  section  by  carrying  the  block  across  the  razor  edge,  the  spring 
D  supplying  the  force.  The  block  is  moved  forwards  for  each  suc- 
ceeding section  b}'  tilting  the  rest  E  razorwards.  The  tilting  is 
accomplished  by  turning  the  screw  F,  which  raises  that  end  of  the 
rest.  F  is  rotated  by  a  catch  G  on  the  lever  C  which  engages  the 
teeth  on  the  large  disc  of  F.  The  catch  G  carries  a  pin  which 
projects  downwards  and  rests  against  a  sector  H.  The  latter  controls 
the   position   of   the   catch    so   that   it    passes    clear   of   the   disc    during 


FIG.  9.     Rocking  Microtome   (modified),   see  text. 
block-holder  (Durham). 


A'   additional 


the  cutting  of  the  section,  i.e.,  down  stroke  of  the  block  A,  and 
also  during  its  up  stroke  until  A  has  passed  clear  of  the  razor. 
The  thickness  of  the  section  is  fixed  by  the  number  of  teeth  through 
which  the  disc  on  F  is  moved  and  this  again  is  determined  by  the 
relative  position  of  H  which  controls  the  catch  G  (each  tooth  equals 
a  thickness  of  4  //).  Adjustments  are  provided  for  regulating  (1)  the 
thickness  of  the  section,  (2)  the  excursions  of  the  rocking  arm.  and 
(3)  the   position   of   the   razor. 

In  the  original  form  of  this  excellent  instrument,  the  razor  is 
carried  in  a  fixed  support.  The  movable  carrier  illustrated  above 
allows,  without  loss  of  rigidity,  of  great  freedom  of  orientation  in 
two  directions,  whilst  the  third  direction  is  obtained  by  turning  the 
brass    thimble    itself.       This    modification,    together    with    the    adjusting 


CUTTING     SECTIONS     OF     FROZEN     TISSUE. 


•<:, 


ring  for  the  cord  and  the  variable  stop  L  were  suggested  by  H.  E.  Durham 
(Proceedings  Physiol.  Soc.,  Jl.  Physiol.,  Vol.  xix.,  p.  xvi.).  The  movable 
carrier  also  permits  different  parts  of  the  razor  to  boused  and  the  ring 
enables  one  to  fix  the  rocking  arm  in  any  position  without  danger  of 
its  slipping. 

Freezing"  and  cutting"  in  gum  mucilage.1  Crystalline  sugar 
1  oz.  in  1  fl.  oz.  water,  gum  mucilage  (1  lb.  gum  accacia  to  8  fl.  oz. 
water)    5  oz.,    water   9  oz.       Mix,    filter,    and    add    thvmol    or    carbolic 


a    lj 


Fig.  10.      Williams'  Ether  Freezing  Microtome.    (Made  by  Swift). 

P  Metal  cap  upon  which  the  tissue  is  placed,  fixed  by  a  vulcanite  collar  in  the 
glazed  platform,  which  is  supported  by  a  pillar  clamped  to  the  table.  S  Atomiser 
actuated  through  F  by  a  foot  blower.  E  Ether  bottle.  C  "Wooden  cap  to  cover  the 
tissue  whilst  it  is  being  frozen.  The  knife  sledge  is  supported  upon  screws  A  for 
lowering  the  razor  after  each  cut,  aa,  for  levelling  the  edge  of  the  razor.  Two  vertical 
screws  bb,  for  adjusting  its  slope  and  two  horizontal  ones  for  fixing  it  in  position. 


1  The  method  of  freezing  by  means  of  ice  and  salt  was  introduced  into  histology 
by  Prof.  W.  Rutherford  in  1872,  Dr.  U.  Pritchard  suggesting  the  use  of  gum  for 
embedding,  to  which  Dr.  D.  J.  Hamilton  (then  of  Edinburgh)  subsequently  made  the 
addition  of  syrup  to  prevent  crystalisation.  Mr.  Bevan  Lewis.  West  Riding  Asylum, 
introduced  ether  as  a  means  of  freezing  in  1876. 


26  EXERCISES    IN    THE    PREPARATION    OF    TISSUES. 

arid,  to  preserve  it.  The  tissue  must  be  completely  freed  of 
alcohol  or  chromium  salts  by  irrigation,  for  24  hours,  in  running 
water ;  it  is  then  left  in  the  gum  for  a  like  period,  or  longer.  The 
more  thorough  the  impregnation  the  better  will  the  mass  cut  when 
frozen.  It  should  then  present  a  cheesy  consistency.  A  microtome 
is  necessary,  such  as  Williams',  in  which  the  freezing  is  accom- 
plished by  means  of  ether.  The  block  of  tissue,  £  to  §  in.  thick, 
is  placed  on  the  brass  plate  with  the  adherent  gum,  is  covered  with 
a  non-conducting  cap,  and  the  ether  spray  is  operated  until  the 
whole  mass  is  completely  solidified.  The  razor  is  carried  in  a  tripod 
frame,  the  front  foot  being  turned  to  regulate  the  thickness  of  the 
section.  Sections  are  then  cut  as  thin  as  possible,  and  are  trans- 
ferred from  the  razor,  upon  which  they  accumulate,  into  a  bowl  of 
water.  Here  they  should  remain  until  cleared  of  gam,  which  may 
necessitate  a  change  of  water.  The  sections  may  be  kept  for  later 
use   in   90 p-c-  alcohol,  provided  well-stoppered   bottles   are   used. 

Embedding  in  celloidin.  Tissue  previously  dehydrated  in  absolute 
alcohol  is  placed  in  a  thin  solution  of  Scherer's  celloidin  in  equal 
parts  of  absolute  alcohol  and  ether,  the  preparation  which  at  first 
floats  at  the  top  sinks  as  the  celloidin  solution  penetrates.  Let  the  fluid 
thicken  to  a  syrupy  consistency  and  set  it  as  follows.  Construct  a 
receptacle  of  blotting  paper,  a  lidless  box  held  together  with  pins,  or 
wrap  a  piece  of  blotting  paper  round  a  cork  so  as  to  leave  a  projecting 
tube  ;  wet  the  paper  with  water,  fill  with  celloidin,  and  put  the 
tissue  in  position,  leave  the  whole  exposed  to  the  air  until  a  film 
forms  on  the  surface,  then  float  on  70 pc-  alcohol  (or  place  in 
chloroform)  until  firmly  set.  The  mass  when  set  should  have  a  nearly 
transparent  and  opalescent  appearance,  and  can  be  preserved  for  future 
use  in  80  P-c-  alcohol. 

When  required  for  cutting,  wash  thoroughly  in  water  (1  hour),  and 
then  immerse  in  gum-freezing  mixture  for  10  to  20  minutes,  and  freeze 
on  a  Williams'  microtome.  It  is  not  desirable  that  the  celloidin  itself 
l>e  frozen,  it  is  enough  if  the  gum  fixes  the  mass  to  the  plate  of  the 
microtome,  as  the  embedding  material  gives  the  necessary  support. 
The  block  held  in  the  hand  can  be  cut  directly  with  a  razor  moistened 
with  alcohol.  It  must  not  be  allowed  to  dry.  The  sections  are  placed 
in  water,  stained,  and  mounted.  To  mount  in  balsam,  clear  with  origanum 
oil    which    does  not  dissolve  celloidin. 


SOLVENTS     AND     STAINS.  27 

Synopsis  of   Treatment  for  Embedding  Tissues  and 
for   Staining"   and   Mounting-   Sections. 


THE    TISSUE    IS    PRESUMED    TO    BE    IN    ABSOLUTE 

ALCOHOL. 

To    embed   in    gum.  To   embed   in    paraffin. 

1.  Wash    24  to  48  hours       1.  Clear  24  to  48  hours,  (b) 
in  running  water. 

2.   Paraffin  1  to  4  hours. 
2.   Pass  into  gum  and  syrup. 
24  hours  to  several  days.  3.   Cut  sections. 

1        . 

3.   Freeze  and  cut  sections.       If  unstained.         If  stained  in 

!  I  bulk. 

4.   Wash  in  water  to  remove  4.  Toluene 

gum.  to  remove  2.       4.  Toluene  or 

cedar  oil.  (b) 

5.   Stain.  5.  Alcohol,   (a) 

/            \  5.  Balsam. 

Mount  in        Dehydrate,  (a)  6.   Stain, 
glycerine  or 

Farrant.              Clear,  (b)  7.   Dehydrate,  (a) 

Balsam.  8.   Clear. 

9.   Balsam. 

(a)  Absolute  alcohol. 

(b)  Toluene. 

A  piece  of  tissue  or  a  section  which  has  been  allowed  to  dry  is  spoilt. 


CHAPTER   III. 

THE    SIMPLE    TISSUES. 

The  methods  of  preparation  are  indicated  by  the  following  abbrevia- 
tions : — p.  =  Preparation,  s.  =  Stain,  c.  =  Method  of  cutting,  G.  =  Gum, 
P.  =  Paraffin.  C.  =  Celloidin.  m.  =  Mounting  fluid  to  be  employed, 
B.  =  Canada  balsam,  F.  =Farrant,  Gl.  =  Glycerin.  The  numbers  refer 
to  paragraphs  in  the  appendix. 

Endothelium.       Omentum   of  a  guinea  pig. 

Treated  with  nitrate  of  silver.  The  piece  of  tissue  in  alcohol  is 
placed  in  water.  Mount  it  in  balsam.  There  are  thicker  portions 
in  which  blood-vessels  run.  Cut  away  the  thickest  parts  by  pressing 
the   edge   of  a   knife   where   3^011   wish   to   sever   the   tissue. 

(//)  Find  on  the  more  delicate  fenestrated  omental  tissue 
the  blackened  outlines  of  the  endothelial  cells,  due  to  the  action 
of  the  silver  salt.  The  cells  adapt  themselves  closely  to  the 
surfaces  which  they  cover.  On  the  non-fenestrated  portions  of 
the  membrane  single  cells  or  small  clusters  of  three  or  four 
cells  more  deeply  stained  than  the  rest  are  met  with  (germinal 
cells). 

Stratified  Squamous  Epithelium.  Detached  cells  from 
the  human  mouth. 

With  your  finger  remove  some  saliva  from  the  inside  of  your 
cheek,    mount   between   glasses. 

Search  (//)  for  large  irregular  cells.  Their  surfaces  exhibit 
slight  ridges,  and  each  has  a  nucleus  surrounded  by  fine 
granules.  The  surface  of  the  cells  is  frequently  covered  with 
coarse  granules  (micrococci).  To  render  the  nucleus  more 
<\ident,  stain  with  magenta  or  Spiller's  purple  by  irrigation. 
Watch    the    staining    taking    place    under    a    low    power,    and 


SQUAMOUS     EPITHELIUM.       MITOSIS.  29 

arrest  its  progress  when  sufficiently  developed  by  running 
water  through  until  the  diffuse  colour  is  removed.  Note 
films    of   precipitated    mucin    and    also    salivary    corpuscles. 

Human   Skin.     V.S.     (p.  11,  s.  22  &.  24,  c.   P.,  m.  B.) 

(L)  Note  the  epidermis  resting  upon  and  filling  up  the 
irregularities  of  the  dermis.  The  epidermis  consists  of  two 
main  layers — the  horny  layer,  externally,  resting  upon  the  rete 
mucosum,  from  which  it  is  sharply  defined  by  the  thin  stratum 
lucidum. 

(II)  In  the  lowest  layers  of  the  rete  mucosum  the  cells  are 
elongated  (germinal  cells),  those  above  being  polygonal,  and 
the  highest  somewhat  flattened  and  of  a  granular  appearance 
(stratum  granulosus).  The  cells  composing  these  layers  are 
united  to  each  other  by  numerous  fine  filamentous  bridges, 
hence  named  "prickle  cells."  A  continuous  system  of  channels 
is  thus  left  between  the  cells  up  to  the  stratum  lucidum  for 
the  percolation  of  lymph  (formation  of  a  blister). 

The  stratum  lucidum  is  formed  by  the  accumulation  of  eleldin 
produced  by  the  cells  of  the  stratum  granulosum.  It  can  be 
detected  accumulating  as  a  thin  layer  between  them,  gradually 
thickening  to  a  continuous  layer  in  which  cell  outlines  are  lost. 
The  cells  of  the  horny  stratum  are  much  flattened  and  com- 
pressed ;  they  are  the  dead  remains  of  the  malpighian  cells,  and 
do  not  exhibit  marked  differential  staining.  Excepting  in  rare 
instances  no  traces  of  nuclei  are  visible.  Osmic  acid  blackens 
the  inner  and    outer  portions  of  the  horny  layer. 

Mitosis.  Nuclear  filaments  from  the  salivary  cells  of  the 
chyronomus  larva.  A  common  inhabitant  of  the  mud  of 
stagnant  waters.  It  is  about  half  an  inch  long,  red  in  colour, 
and  progresses  by  jerky  unbending  from  a  C  shape. 

Pull  the  head  off  with  forceps,  cover  in  normal  saline,  irri- 
gate   with    an    aqueous    2  pc    solution    of    methyl    green    until 


30  THE    SIMPLE    TISSUES. 

stained.  Wash  with  water  acidulated  with  acetic  acid,  and 
replace  the  latter  by  glycerin.  (H)  Find  the  nuclei  of  the 
large  salivary  gland  cells,  and  in  them  the  coiled  and  obvious 
nuclear  filamena. 

V.S.  Jaw  of  young  newt.  (p.  9,  s.  22,  c.  P.,  m.  B.)  Find 
amongst  the  lower  epidemic  cells  nuclei,  showing  the  convolute 
and  aster  stages,  these  are  the  most  easy  of  recognition. 

Columnar  epithelium.  Y.S.  Stomach  of  an  adult  cat. 
(p.  3,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Recognise  the  ducts  of 
the  gastric  follicles,  wide  and  somewhat  oval  recesses  into  which 
the  tubular  gastric  glands  open.  (H)  Their  cavities  as  well  as 
the  general  surface  of  the  stomach  are  covered  with  this  variety 
of  epithelium.  The  cells  are  taller  than  they  are  broad,  are 
enclosed  in  a  thin  cell  membrane,  the  contents  of  whick  are  clear 
and  traversed  by  a  delicate  cytoplasmic  network,  the  nucleus 
is  placed  near  the  attached  end.  Under  certain  methods  of 
treatment  intercellular  bridges  are  seen  between  them  (Carlier). 

Epithelium  of  the  small  intestine.  V.S.  Small  intestine 
— cat,  dog,  or  newt.      (p.  11,  s.  22  &  24,  c.  P.,  m.  B.) 

(L)  Find  the  villi,  column-like  projections  of  the  mucosa  into 
the  cavity  of  the  gut.  On  their  surface  (II)  a  single  layer  of 
columnar  nucleated  cells,  with  a  marginal  hem  on  their  free 
surface.  This  is  the  sectional  appearance  of  the  end  plate, 
which  is  probably  composed  of  short  prismatic  rods  set  close 
together  on  the  end  of  the  cell.  Sections  of  the  cells  showing  the 
plates  in  surface  view  are  to  be  sought  for,  they  appear  as  polygonal 
ureas  with  fine  punctate  markings.  In  the  intestine  of  the  cat, 
among  the  columnar  cells,  Whatney's  buds  may  frequently  be 
met  with,  these  consist  of  zones  of  cells  arranged  transversely 
around  the  villi,  which  in  sectional  view  appear  as  clusters  of 
cells    tapering    towards    their   free   ends.       Whatney    described 


riU.\Ti:i>     EPITHELIUM. 


31 


these  as  cells  in  a  .state  of  proliferation.  Chalice  cells,  unicellular 
mucous  glands,  are  numerous  amongst  the  columnar  cells.  Some 
are  full  of  a  transparent  secretion  (mucigefa),  others  empty  and 

collapsed.  The  former  are  like  footless  wineglasses.  Leucocytes 
occur  frequently  between  the  cells. 

Ciliated  Epithelium.  Trachea  of  a  child.  T.S.  (p.  8, 
s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  the  inner  layer.  (II)  The 
free  surface  is  covered  with  columnar  ciliated  epithelium,  the 
lower  layers  of  cells  are  pear-shaped  or  rounded,  and  rest  upon 
a  well  defined  basement  surface.  The  epithelium  is  renewed  by 
the  proliferation  of  the  lowest  cells. 

Isolated  ciliated  cells  from   the  pharynx  of  the  frog  (p.  24  to 

4^  hours  in  33  pa  alcohol,  coloured  by  Picrocarmine  (Ranvier) 
m.  Gl.)  (H)  Irregular  wedge-shaped  nucleated  cells,  the  free 
ends  of  which  are  tufted  with  cilia  implanted  in  a  marginal  zone. 
Chalice  cells,  some  full  of  granules  (mucigen.)  stained  yellow, 
others  empty,   occur  in  considerable  numbers. 

Ciliary  action.1  Open  a  mussel  by  cutting  through  the  hinge, 
Fig.  11,  then  pass  the  knife  between  the  shells  to  sever  the  adductor  A  and 
soft  parts.  The  thick  border 
of  the  mantle  lies  within  the 
long  side  of  the  shell.  It 
retracts  on  being  touched  if 
the  mussel  is  alive.  Lying 
upon  the  mantle  in  a  double 
layer  is  the  gill.  Cut  out  a 
small  piece  of  the  gill  witli 
scissors,  place  it  on  a  cover- 
glass  in  a  drop  of  fluid 
from  the  mussel,  and  separate  the  two  layers.  Invert  the  preparation 
(hanging  drop)  upon  a  cell  formed  of  three  thicknesses  of  blotting  paper, 
*ut  so  as  to  fit  the  slide,  and  with  a  central  aperture  §  inch  in  diameter, 
and  moisten  with  salt  solution.    Fig.  12. 


Fie.  n. 


A  scraping  from  the  pharynx  of  the  frog  diffused  in  normal  saline  answers  very  well. 


32 


THE    SIMPLE    TISSUES. 


(L)  The  gills  consist  of  bars  with  clubbed  free  ends,  along 
their  edges  recognise  the  movements  due  to  ciliary  action.  (//) 
On  the  edges  of  the  bars  the  cilia  are  seen  in  full  face  curving 
towards  or  away  from  the  observer,  whilst  at  the  free  unbroken 


Fig.  12. 

ends  they  are  seen  in  side  view.  Note  the  direction  of  the 
movement  and  the  manner  in  which  the  cilia  bend.  The  cur- 
rents produced  are  indicated  by  the  movements  of  the  floating 
particles  in  the  fluid.  Detached  pieces  of  gill  occur  rotating 
under  the  influence  of  their  cilia. 

Effect  of  chloroform  or  ether.  Raise  the  cover,  introduce  a  small  drop 
of   the  reagent  into  the  cell,  and   replace  the  cover. 

Watch  the  gradual  slowing  and  ultimate  arrest  of  the  move- 
ment. As  the  movement  slows  observe  the  way  in  which  the 
individual  cilium  bends,  it  curves  from  the  tip  downwards  and 
the  extension  takes  place  in  the  converse  direction.  If  the 
action  of  the  reagent  has  not  been  excessive  motion  can  be 
restored  by  removal  of   the  reagent. 

Free  the  cell  of  its  gaseous  contents,  lift  off  the  cover,  remove  the 
paper  cell  with  forceps,  and  after  rinsing  the  latter  in  water  replace 
them  in  position. 

The  cilary  action  will  slowly  return.  This  is  an  instance 
of    the  action  of  an  anaesthetic  upon  living  protoplasm. 

Effect  of  heating-.  Using  the  same  preparation  and  omitting  the 
paper  cell,  place  it  on  the  hot  stage  upon  the  stage  of  the  microscope.  Tnrn 
1 he  end  of  the  tin  plate  which  projects  beyond  the  stage  slightly  upwards 


MOT    STAGE.— CONNECTIVE     TISSUK. 


33 


and  sot  the  flame  of  the  bunsen  burner  under  this  part.  Guard  against 
over-heating  by  periodically  testing  the  plate  between  the  stage  and 
flame  with  your  finger. 

The    ciliary     movement    quickens.        If    the    temperature    be 
sufficiently    elevated    the    motion    will    cease    {heat  stiffening). 


FlG.  VS.  Hot  stage.  A  plate  of  tin  insulated  from  the  stage  of  the  microscope 
by  a  piece  of  blotting  paper  B,  S  the  slide  through  which  is  seen  the  central 
opening  in  the  stage,  H  line  along  which  the  plate  is  bent  up  ;  the  flame  is  placed 
under   this   end. 

Secreting   Epithelium.     See  digestive  tract. 

Connective  tissue  proper.     Areolar  tissue.     Spread  a  small  piece    Q 
of   the   subcutaneous   tissue    of    a  mammal  (rabbit  or  rat),   by  means   of 
needles,  on  a  dry  slide,  into  as  thin  a  film  as  possible  (semi  desiccation), 
breathing  upon  the  tissue  from  time  to  time  to  obviate  drying.     Moisten 
with  normal  saline  and  cover. 

Recognise  the  following  :  (a)  White  fibres,  transparent  wavy 
bundles  exhibiting  delicate  longitudinal  markings,  (b)  Elastic 
fibres,  fine  straight  filaments,  some  of  great  tenuity  with 
distinct  though  infrequent  branchings,  broken  ends  tend  to 
curl  up  into  irregular  coils.  Their  higher  refractive  index 
gives  them  a  sharp  outline  when  in  focus.  (c)  Connective 
tissue  corpuscles,  difficult  to  detect  in  the  rabbit,  more  easily 
seen  in  the  rat  and  guinea  pig. 

Effect  of  acetic  acid.     Irrigate  with  1  p-c-  solution. 

Observe  that  the  white  fibres  are  rendered  more  transparent 
and  that  they  swell.  Constrictions  may  be  made  out  in  places 
where  a  ring-like  filament  encircles  a  fibre.  The  elastic  fibres 
now  appear  distinct,  and  the  corpuscles  or  at  least  their  nuclei 
become  evident. 


34  THE    SIMPLE    TISSUES. 

Stain  the  cells.  Irrigate  first  with  water  to  remove  the  acid, 
and  then  stain  with  hoemalum.  When  the  staining  is  completed  wash 
with  water,  and  after  removal  of  the  cover  dehydrate,  clear   and   m.  B. 

Cell  spaces  in  areolar  tissue.  Treat  a  thicker  film  obtained 
as  above  with  a  drop  of  AgNOs  sol.  1  p-c-  and  expose  it  to  bright 
sunlight,  in  15  to  20  minutes  a  brown  colour  will  develop. 

Examine  (ff),  find  the  irregular  uncoloured  cell  spaces  on 
a  brown  ground   (Schafer). 

As  soon  as  these  are  evident,  uncover  and  rinse  the  preparation 
carefully   with  water,   dehydrate  and  mount  in  balsam. 

Another  preparation  to  bring  the  elastic  fibres  into  view  can  be 
made  by  irrigating  a  fresh  specimen  with  Spiller's  purple,  which 
stains   them    violet.     (Roseanilin  nitrate  stains  them  red — "Schafer.") 

Tendon.     Fresh  tendon.      Rat's  tail  teased  in  normal  saline. 
(L)  Consist  of  bundles  of  white  fibrous  tissue  upon  which  (H) 
rows  of  cells  are  indistinctly  recognisable. 

Effect  of  acetic  acid.  The  tissue  clears  and  swells  and  the 
cells  come  into  view  arranged  in  rows,  each  with  its  nucleus. 
Very  little  elastic  tissue  is  visible. 

Stain  with  hcemalum  as  above. 

Treat  a  fresh  portion  in  AgNO.  solution  for  cell  spaces  and  outlines 
of  the  epithelial  tendon  sheath. 

Fresh  tendon  from  the  frog's  foot  may  be  obtained  by  seizing  the  tip 
of  a  toe  with  strong  forceps,  and  pulling  a  tendon  out  of  the  foot 
with  it. 

Rat's  tail.  T.S.  (p.  14  (b),  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  the 
tendon  bundles  lying  in  grooves  around  the  vertebrae.  Recognise 
the  sheaths  of  fibrous  tissue  which  surround  them,  and  (H)  the 
tendon  cells  branched  and  deeply  stained,  which  are  dis- 
tributed between  the  tendon  fibres. 

Rat's  tail.  L.S.  (Prepared  as  above.)  Note  the  regular 
arrangement  of  the  tendon  cells  into  rows,  the  position  of 
their  nuclei,  and  the  spread  of  their  protoplasm.       The    proto- 


TENDON.  —  ELASTIC     TISSUE.  35 

plasm  often  exhibits  a  ridge-like  marking  at  the  thickest  part, 
which  extends  along  the  row,  this  is  due  to  the  moulding  of 
the  cells  by  neighbouring  bundles,  and  is  known  as  Boll's 
stripe. 

Tendon  of  a  large  mammal.  T.S.  (p.  formol  4pc  and  acetic 
acid  lp%  each  1  week,  c.  G.,  s.  19,  m.  F.)  The  tendon  cells 
are  much  less  numerous  proportionately,  than  in  the  rat's  tail, 
and  are  not  so  regular  in  their  arrangement.  Their  number 
is  largely  a  question  of  age.  In  young  tendons  they  are  much 
more  numerous.  Fine  elastic  fibres  are  present,  and  are  to  be 
recognised  as  small  dots  in  the  white  fibrous  substance. 

Yellow  fibrous  or  elastic  tissue.  Lig amentum  Nucha?, 
Ox.,  (p.  2  (d),  s.  19,  m.  F.). 

Tease  some  of  this  tissue  digested  in  acetic  acid,  it  is  more  readily 
dissociated  than  the  fresh  tissue,  as  the  white  fibrous  tissue  has  been 
softened   by  the  acid. 

(H)  The  fibres  branch  and  form  a  network  of  elongated 
mesh,  the  coarser  fibres  occasionally  exhibiting  transverse 
linear  perforations.  On  the  concavities  of  bent  fibres  slight 
transverse  corrugations  may  be  perceived,  these  are  indicative 
of  a  surface  membrane.  No  nuclei  are  visible  in  or  imme- 
diately connected   with  the  fibres. 

Ligamentum  Nucha?  (p.  2  {d),  c.  G.,  s.  19,  m.  F.).  L.S.  The  Elastic 
tissue  fibres,  stained  yellow  by  the  picric  acid,  are  surrounded 
by  white  fibrous  tissue  stained  pink,  in  the  latter  nuclei  are 
recognisable,  but  none  in  the  yellow  tissue.  Observe  the 
branching   of  the  fibres. 

T.S.  (H)  Note  the  yellow  fibres  cut  across,  forming  irregular 
clusters,  embedded  in  the  pink  white  fibrous  tissue  as  before. 
In  the  large  arteries  elastic  tissue  occurs,  resembling  the 
above,   but  of  finer  texture.      See  later. 


36  THE    SIMPLE    TISSUES. 

Retiforni  or  adenoid  tissue,  S.  of  a  Lymph  Gland  of 
an  ox  or  sheep. 

Injected  interstitially  with  a  0*25  p-c-  sol.  AgNO..,  hardened  in 
alcohol,  cut  by  freszing  in  gum,  and  stained  with  Hrematoxyline, 
m.   B. 

(L)  Find  the  lymph  sinus  in  the  outer  part  of  the  section, 
(H)  recognise  the  delicate  branching  tissue  extending  across 
the  spaces,  on  this  find  endothelial  outlines  and  the  nuclei 
belonging  to  the  cells.  The  supporting  material  is  connective 
tissue  (gelatigenous  tissue).  Numerous  cells,  the  lymph  cor- 
puscles, are  distributed  throughout  the  meshes  of  the  branching 
material,  very  closely  crowded  in  the  follicular  tissue  outside 
the  sinuses.  These  cells  are  identical  with  the  white  corpuscles 
of  the  blood  and  originate  from  the  epitheliod  elements  upon 
the   branching   tissue.. 

Take  a  section  of  the  fresh  gland,  cut  by  freezing,  shake  it  vigorously 
in  normal  saline  in  a  test-tube  for  a  few  minutes.  Mount  the  fragments 
in  Farrant's  Solution,  tinged  with  Picrocarmine.  The  retiform  tissue, 
now  cleared  of  the  corpuscles,  will  show  its  finer  branchings. 

g  Fatty  Tissue.  Epiglottis  of  kitten  or  young  human  skin. 
V.S.  (p.  3,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  fat  cells  stained 
black  by  the  osmic  acid.  (//)  Trace  the  accumulation  of  fat  in 
the  cells,  first  as  small  granules  in  the  peripheral  cells,  growing  to 
mulberry-like  masses  in  others,  and  ultimately  to  a  single  large 
globule  which  distends  the  cell  uniformly.  Fatty  tissue  will 
be  of  common  occurrence  in  the  tissues  and  organs  studied  in 
the  sequence. 

After  treatment  with  essential  oils  for  the  purpose  of  embedding  in 
paraffin  the  cell  envelopes  arc  usually  found  empty,  their  contents  having 
b.;en  removed  by  the  solvent.  Fatty  tissue  mounted  in  glycerine  exhibits 
crystals  of  trimargarine  and  tristearine,  triolein  only  remaining  fluid 
at  ordinary  temperatures. 


MUCOUS    TISSUE.— CARTILAGE.  37 

Mucous  tissue.    Umbilical  cord,  man.   T.N.  (p.  12,  c.  G.,  s.  19, 

m.  F.)  (L)  The  section  is  rounded;  in  it  observe  three  circular 
structures,  two  arteries  and  a  vein  surrounded  by  connective 
tissues  of  an  open  texture.  (//)  In  the  latter  are  found  branch- 
ing cells  with  delicate  irregular  processes  lying  in  a  transparent 
matrix  (mucinous  substance)  which  exhibits  a  good  deal  of 
fibrillation  in  its  later  stages  of  growth. 

Cartilage.  Hyaline  cartilage.  Young  costal,  (p.  2  [d)}  c.  G., 
s.  19,  m.  F.)  (L)  Externally  there  is  the  fibrous  perichondrium 
inside  this  the  substance  consists  of  matrix,  apparently  struc- 
tureless, in  which  the  cartilage  cell-spaces,  irregularly  fusi- 
form in  shape,  are  distributed.  In  the  hardened  specimen  the 
cells  do  not  always  completely  fill  the  spaces  in  which  they 
lie,  as  they  are  usually  somewhat  shrunken.  Towards  the 
periphery,  close  to  the  perichondrium,  the  spaces  often  com- 
municate with  each  other.  In  the  interior  the  older  capsules 
do  not  show  these  connections.  The  transition  of  the  connective 
tissue  cells  of  the  perichondrium  into  those  of  the  cartilage 
should  be  noted. 

Cartilage  of  cuttlefish,  (p.  2  (rf),  c.  G.,  s.  19,  m.  F.)  (7/)  The  cell 
spaces  occur  in  clusters,  and  from  these  fine  branching  channels 
run  outwards  to  communicate  with  other  groups  and  establish 
direct  communications  with  their  spaces. 

Adult  costal  cartilage  Human.  (p.  2  (d),  c.  G.,  s.  19,  m.  F.) 
The  cells  are  in  clusters  immediately  around  which  the  matrix 
is  hyaline,  outside  this  fibrillated,  and  in  the  aged  and 
unsoftened  tissue   contains  lime  salts. 

Articular  cartilage.  V.S.  (p.  2  (d),  c.  G.,  s.  19,  m.  F.)  The  film 
of  cartilage  which  covers  the  articulars  end  of  bones  is  hyaline. 
Near  the  free  surface  the  cells  are  flattened,  deeper  down  they 
are  oval.     The  matrix  close  to  the  subjacent  bone  is  fibrillated 


38  THE    SIMPLE    TISSUES. 

and  in  the  recent  state  calcareous.  An  irregular  line  separates 
the  cartilage  from  the  subjacent  bone.  Shallow  depressions, 
recesses  communicating  with  the  medullary  cavity  and  containing 
blood  vessels,  are  visible ;  these  become  filled  later  with  boss-like 
deposits  of  bone. 

White  Jibro-cartilage.  S.  Intervertebral  disc  (p.  11,  c.  G.,  s.  19, 
m.  F.).  (Z)  The  arrangement  into  concentric  layers  is  most 
distinct  at  the  periphery.  (H)  A  gelatinous,  fibrous-looking 
material,  with  scattered  small  clusters  of  cells  in  thin  envelopes. 
In  the  centre  of  the  disc,  if  included  in  the  section,  is  seen 
an  agglomeration  of  various  sized  cells  with  distinct  capsules, 
and  little,  if  any,  interstitial  substance.  This  is  a  relic  of 
the  notochord,  and  the  nearest  approach  to  parenchymatous 
cartilage    in    the    human    body. 

Interarticular  cartilages  have  the  same  structure,  but  the 
laminar  arrangement  is  not  present,  the  fibres  of  the  matrix 
being    more   interlaced. 

Spongy,  yellow  o'r  elastic  cartilage.  T.S.  Ejriylottis  (p.  3,  c.  G., 
s.  19,  m.  F.).  (L)  Externally  stratified  squamous  epithelium 
under  this  connective  tissue,  pink,  in  it  are  mucous  glands.  The 
cartilage,  surrounded  by  fibrous  perichondrium,  is  perforated 
with  large  apertures  filled  with  fibrous  tissue  containing  blood 
vessels.  The  cartilage  is  tinted  yellow.  At  its  edges  (//) 
observe  the  elastic  fibres  passing  from  perichondrium  to  matrix, 
and  which  beginning  as  fine  filaments  quickly  further  in  pass 
into  a  close,  spongy  structure  enclosing  the  cell  spaces.  These 
contain    the    nucleated    cartilage    cells. 

Arytenoid  cartilage,  (p.  11,  c.  G.,  s.  19,  m.  F.)  (L)  Fibrous 
nature  of  the  tip.  (//)  The  matrix  of  the  cornu  is  yellow 
fibro-,   passing   into  the   hyaline   cartilage   towards   the   base. 


CHAPTER    IV. 
BONE. 
Bone.      (T.S.)     Dried    long   bone    of   man. 

Slices  cut  with  a  saw,  ground  thin,  polished  and  mounted  in 
halsam. 

(L)  The  matrix  of  adult  bone  consists  of  thin  layers 
or  lamellce  composed  of  white  fibrous  tissue  in  a  calcified 
ground-substance.  The  fibres  of  alternate  lamellae  are  arranged 
at  different  angles,  and  the  lamellation  is  most  easily  recog- 
nised where  one  set  is  cut  transversely.  The  general 
arrangement  can  be  made  out  by  observing  the  disposition 
of  the  lacunce.  These  are  oval  flattened  spaces  in  the 
lamellae,  the  long  dimensions  of  which  correspond  to  the 
planes  of  the  latter,  and  which,  in  the  absence  of  soft  parts, 
as  in  this  case,  are  filled  with  air,  and,  in  consequence, 
appear  black.  (L)  The  outer  lamellae  are  set  parallel  to 
the  surface  of  the  shaft  (periosteal  lamella?).  Further  in 
they  form  concentric  Haversian  systems,  each  of  which  has 
in  its  centre  a  Haversian  canal,  the  latter  usually  appears 
opaque,  through    being    filled    with   detritus. 

Among  the  periosteal  lamella?  and  between  the  systems 
are  found  here  and  there  irregular  rounded  openings,  the 
Haversian-  spaces,  which  have  been  eroded  for  the  deposition 
of  new  systems.  Where  the  cavity  is  still  empty  its  outline 
is  pitted.  In  others  lamellae  are  found  lining  them  in  greater 
or  lesser  numbers,  the  central  cavity  being  proportionally 
diminished. 


40  BONE. 

On  the  inner  surface  of  the  shaft  are  the  cancellous  lamella?, 
forming  a  spongy  network  of  greater  richness  the  nearer 
the  origin  of  the  section  is  to  the  end  of  the  bone. 
(H)  The  Haversian  canals  and  the  lacunae  intercommunicate 
freely  by  means  of  fine  channels,  the  canaliculi,  and  the  latter 
form  at  the  periphery  of  the  systems  closed  loops  "  recurrent 
canaliculi.''''  Search  for  well-defined  lamelhe  in  the  Haversian 
systems. 

Shaft  of  long  bone.  L.S.  Similarly  prepared.  (L)  The 
Haversian  canals  form  a  longitude  system  of  anastomosing 
channels,  which  open  both  upon  the  outer  and  inner  surfaces 
of  the  bone. 

(II)  The  grouping  of  the  lamellae  into  systems  is  less  recog- 
nisable. The  Haversian  spaces  may  be  traced  in,  favour- 
able positions  to  be  expansions  of   Haversian  canals. 

Cranial  bones.  The  plate-like  bones  (L)  exhibit  an  inner  and  an 
outer  layer  [table)  of  dense  osseous  material,  separated  from  each  other 
by  cancellous  tissue  (Dip/oe). 

Sharpey's  fibres.  In  the  outer  lamellae  of  dense  bone  fibres 
occur  which  traverse  them  vertically  from  the  surface.  These  are  some 
of  the  more  obvious  fibres  of  periosteal  origin  which  have  become 
included  in  the  lamellre  during  the  growth  of  the  latter.  Some  of  them 
are  elastic  fibres. 

Softened  bone.  (p.  14,  c.  G.,  s.  19,  m.  F.)  The  lime  salts  are 
removed  and  the  soft  parts  preserved.  (I)  Externally  is  the 
/'xriosteum,  dense  in  its  outer  (fibrous)  layer,  and  more  open 
in  its  inner  (osteognetic)  layer.  The  latter  is  attached  to  the 
subjacent  bone  itself.  These  subdivisions  are  more  striking  in 
the  growing  stage.  (//)  Observe  the  fusion  of  the  periosteal 
fibres  to  the  bone  matrix,  especially  where  the  tendons  are 
inserted.  The  lacunae,  less  sharply  defined,  each  accommodate 
a   bone   corpuscle.      The   canaliculi    being    filled    with    fluid,    do 


GROWING     BONE.  41 

not  show  clearlv  beyond  their  origins  in  the  lacunae.  The 
Haversian  canals  are  incompletely  filled  by  blood  vessels. 
around  which  are  perivascular  lymph  spaces.  The  latter  usually 
contain  some  fat. 

The  canaliculi  open  into  the  lymph  spaces,  their  system 
becoming  thus  continuous  with  that  of  the  lymphatics.  Other 
features  recognised  in  the  dry  preparation,  should  he  sought 
tor. 

Head  of growing long bone*  V.S.  Distal  end  of  femur.  Kitten 
or  rabbit,  (p.  8,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Observe  the  cartila- 
ginous epiphysis  (possibly  with  cancellous  tissue  in  its  interior), 
find  where  it  is  implanted  in  the  shaft,  and  note  that  the  arrange- 
ment of  the  cartilage  corpuscles  in  the  latter  part,  is  in  rows 
parallel  to  the  length  of  the  bone.  This  is  the  lifting  zone. 
On  its  inner  aspect,  the  substance  of  the  epiphysis  passes  into 
a  network-like  material,  the  primary  cancellous  tissue,  which 
diminishes  in  quantity  the  further  into  the  shaft  it  is  traced. 
Examine  (H)  the  lifting  zone.  The  outer  longitudinal  rows 
of  corpuscles  form  the  zone  of  proliferation,  deeper  in,  the 
corpuscles  increase  in  size,  the  intervening  matrix  is  diminished 
and     has    a    faintly    granular    appearance,    in    the    unsoftened 

1  The  long  bone  is  originally  laid  down  as  a  cartilaginous  rod.  This  rod  is  cut 
across  by  the  formation  of  the  primary  medullary  cavity,  thus  yielding  two  cartilagi- 
nous heads  or  epiphyses.  These  are  implanted  in  the  ends  of  the  tubular  shaft  which 
now  becomes  evident.  The  cartilaginous  heads  slowly  recede  in  an  axial  direction 
from  the  shaft  by  changes  in  the  cartilage  itself,  and  the  shaft  lengthens  by  growth 
nt  its  ends,  and  thus  keeps  pace  with  the  receding  epiphyses.  Later  the  cartilaginous 
heads  themselves  become  eroded  and  replaced  by  cancellous  tissue  which  first  gives 
in  them  the  appearance  of  the  so-called  centres  of  ossification.  The  lifting  of  the 
heads  occurs  through  the  changes  in  the  inner  (sub-epiphysal)  portion  of  the 
cartilage,  i.e..  that  portion  which  is  embraced  by  the  end  of  the  shaft,  and  which 
may  therefore  be  called  the  lifting  cartilage.  Meanwhile,  the  shaft  is  increasing 
in  thickness  by  snb-periostial  deposition,  and  it  is  maintained  at  a  proportionate 
thickness  by  the  resorption  of  material  from  its  inner  surface.  All  bone  formation  is 
due  to  osteoblasts  and  resorption  to  osteoclasts,  both  of  which  are  corpuscular 
constituents  of  the  periosteum,  or  of  its  continuation  inside  the  medullary  cavity, 
the  Endosteum.  All  bones  prefigured  in  cartilage,  whatever  their  shape,  undergo 
similar  changes,  modified  to  suit  their  particular  case.  Bones  not  or  only  partially 
preceded  by  cartilage  are  developed  from  periosteum,  i.e.,  cranial  bones,  clavicle,  &c. 


4'2  BONE. 

condition  it  is  calcined,  zone  of  increase.  Farther  towards 
the  medullary  cavity,  the  cells  become  replaced  by  a  vascular 
prolongation  of  the  marrow,  zone  of  invasion,  and  the  inter- 
vening matrix  projects  bare  into  the  medullary  cavity,  where 
it  soon  gains  a  covering  of  osseous  material.  The  latter  is 
elaborated  by  the  osteoblasts,  which  are  seen  in  great  numbers 
upon  its  surface.  The  honeycomb  of  cartilaginous  matrix  thus 
left  standing  and  shrouded  in  bone,  constitutes  the  temporary 
cancellous  tissue.  Resorption  of  this  cancellous  material  now 
occurs  by  the  osteoclasts,  large  many-nucleated  corpuscles, 
which  are  found  upon  the  surface  of  the  network,  or  seated 
in  shallow  depressions  of  their  own  excavation.  The  reduction 
of  the  cancellous  tissue  is  thus  brought  about.  It  serves  the 
temporary  purpose  of  a  fixed  point  for  the  pushing  off  of  the 
cartilaginous  head.  Note  the  tapering  edge  of  the  shaft 
outside  the  cartilage,  and  the  strong  fibrous  periosteum  which 
surrounds  it  and  the  cartilage  in  this  region.  In  the  recess 
formed  by  the  projection  of  the  condyles,  note  that  the  surface 
of  the  shaft  under  the  periosteum  presents  excavations  in 
which  there  are  osteoclasts.  The  osteoclasts  are  acting  as 
bone  resorbers  {resorption  area). 


■u 


ic\  Shaft  of  yrowing  bone  of  a  young  mammal.  T.S.  Shaft  (kitten), 
(p.  8,  s.  22  &  24,  c.  P.,  m.  B.)  (Z)  The  two  layers  of  the  periosteum 
are  clearly  defined,  and  the  richness  in  corpuscular  elements  of 
the  osteogenetic  layer  is  striking  when  compared  with  that 
of  the  adult  structure.  The  bone  is  open  in  texture.  Instead 
of  small  canals  there  are  large  spaces,  and  Haversian  systems 
are  absent.  The  surface  of  the  bone  is  covered  by  a  layer 
of  osteoblasts,  excepting  in  those  localities  in  which  osteoclasts 
occur.  Remains  of  marrow  on  the  inner  surface  are  usually 
recognisable.  (//)  The  osteogenetic  layer  of  the  periosteum 
presents    loose   strands    of   connective    tissue,   which    pass    from 


(J  ROWING  BONE. MARROW.  43 

it  to  the  bone  matrix.  Where  the  spicular  outgrowths  of 
the  shaft  are  taking  place,  these  fibrous  connections  are 
evident,  and  numerous  osteoblasts  surround  and  lie  amongst 
their  filaments.  Osteoblasts  are  included  at  regular  intervals 
in  the  newly-deposited  bony  matrix  (bone  corpuscles).  Observe 
the  gradual  inclusion  of  the  lacunae,  and  the  formation  of 
the    canaliculi   as    the   deposition    of    bone   proceeds. 

Ossification  of  the  head  takes  place  by  a  process  closely 
resembling  that  which  occurs  in  the  lifting  zone  below.  The 
cartilage  capsules,  becoming  enlarged  by  proliferation  of  the 
cartilage  corpuscles  and  absorption  of  the  intervening  matrix, 
are  invaded  by  marrow;  temporary  cancellous  tissue  is  formed, 
which  is  ultimately  replaced  by  erosion  and  surface  deposition 
of  bone  until  only  a  thin  layer  remains  upon  the  surface  of 
the  head  as  articular  cartilage  and  between  the  shaft  and 
head  as  the  disc  of  lifting  cartilage.  The  final  union  of 
the  head  and  shaft  takes  place  when  the  lifting  cartilage 
disappears. 

Marrow.  Carefully  break  up  some  red  marrow  in  normal 
saline.  (H )  Find  the  following  :  —  (a)  Marrow  cells  proper 
(Kolliker),  a  little  larger  than  leucosites  and  with  a  large 
round  nucleus  or  sometimes  two.  (b)  Erythroblasts  smaller 
than  the  last,  nucleated,  and  having  a  reddish  tinge  in  the 
fresh  state.  (c)  Large  many-nucleated  cells — giant  cells 
(Myeloplaxes  of  Robin),  these  are  in  many  cases  osteoclasts. 

Red  marrow.  S.  (p.  3.,  s.  22  &  24.,  c.  P.,  m.  B.)1  Good 
preparations  will  be  obtained  in  young  bones.  (H)  Find  the 
myeloplaxes  and  the  other  two  varieties  of  cells.  The  sec- 
tions should  be  very  thin.  Notice  the  wide  thin-walled  blood 
vessels   in  its   substance. 

1  Or  marrow  treated  in  Ranvier's  alcohol  and  picrocarmine  and  mounted  in  gly- 
cerin jelly.     Dried  film  preparations  treated  like  blood  films  yield  good  preparations. 


CHAPTER  V. 

DEVELOPING  TOOTH. 

Tooth.  Adult,  in  the  jaw  of  a  cat.  (p.  8,  c.  G.,  s.  19,  ra.  F.)  (L) 
The  tooth  consists  mainly  of  dentine.  That  part  which  projects 
beyond  the  gum  is  the  crown,  and  is  covered  with  enamel  (here 
removed).  From  the  neck  downwards  the  fang  is  implanted 
in  the  alveolar  cavity,  and  is  covered  by  the  crusta  petrosa,  a 
thin  layer  of  bone.  The  alveolar  walls  are  formed  by  the  bone 
of  the  jaw.  Between  the  jaw  and  the  fang  is  the  dense  fibrous 
peridental  tissue.  In  the  middle  of  the  dentine  is  the  pulp 
cavity  which  communicates  with  the  exterior  through  an 
aperture  at  the  apex  of  the  fang  and  through  which  blood 
vessels  and  nerves  enter.  (H)  The  dentine  is  traversed  by 
numbers  of  minute  canals,  the  dentinal  tubules.  Commencing 
on  the  surface  of  the  pulp  cavity  they  radiate  outwards, 
dividing  occasionally.  Minute  secondary  offshoots  leave  them, 
most  numerously  near  to  and  constituting  their  peripheal 
terminations.  A  zone  of  inter-globular  Spaces,  better  seen  in 
dry  preparations  is  found  in  the  outer  part  of  the  dentine, 
especially  in  the  region  of  the  neck.  They  derive  their  name 
from  the  characters  of  their  outlines.  The  crusta  petrosa 
increases  in  thickness  towards  the  apex  of  the  fang ;  in  it  are 
found  lacunae,  the  matrix  being  the  same  as  that  of  bone. 
The  fibres  of  the  peridental  membrane  are  embedded  in  it, 
and  form  a  tendinous  attachment  to  the  alveolar  wall.  The 
pulp,  an  open  connective  tissue  texture,  contains  blood  vessels, 
lymphatics,  and  nerves;  the  odontoblast  cover  its  surface  and 
lie  in   contact    with  the    dentine,   from  them   delicate   processes 


DEVELOPING     TOOTH.  45 

Tarries  fibres  pass  into  the  tubules.  They  are  better  seen  in 
the  developing  tooth.  Find  tubules  cut  transversely  and  note 
the  .appearance  of  a  tubular  wall. 

Young  tooth.  Early  stage.  T.S.  Fore  part  of  head  of 
embryonic  rat.  (p.  14  (6),  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  the 
cavity  of  the  mouth,  and  the  epithelium  with  stained  nuclei 
which  lines  its  surface.  iSfote  in  each  jaw,  on  each  side,  the 
cU  ntal  groove  tilled  with  epithelium,  from  which  flask-shaped 
prolongations  project  inwards,  the  future  enamel  organs.  Each 
of  these  has  a  conical  recess  on  its  inner  aspect  which 
accommodates  the  dental  papil/a.  The  latter  at  this  stage 
shows  only   as  a  collection  of  nucleated  cells. 

Young  tooth.  Later  stage.  V.T.S.  Jaw  of  kitten,  (p.  14  (b), 
s.  22  &  24,  c.  P.,  ra.  B.)  [L)  The  enamel  organ  developed  from 
the  flask-shaped  mass  of  cells,  is  larger  and  exhibits  epithelium 
on  its  surface  only,  the  interior  of  the  organ  being  occupied  by 
delicate  branching  tissue.  The  surface  cells  are  squamous  in 
single  layer,  excepting  over  the  conical  recess,  which  fits  upon 
the  papilla  ;  here  there  are  several  layers,  the  most  superficial 
of  which  is  columnar.  These  columnar  cells  produce  the  enamel 
prisms.  The  enamel  forms  a  thin  layer,  thickest  at  the  apex 
of  the  cone  and  tapering  towards  the  edges.  It  consists  of 
enamel  prisms  set  side  by  side.  Where  these  are  detached 
from  the  columnar  cells  processes  from  the  latter  should  be 
sought  for,  which  have  been  wrenched  out  of  the  enamel  prisms, 
in  which  they   were  embedded. 

In  immediate  contact  with  the  enamel  is  a  layer  of  dentine, 
forming  a  conical  cap  to  the  papilla,  the  tissue  of  the  papilla 
or  pulp  of  the  tooth  consists  of  a  prominent  layer  of  odonto- 
blasts next  to  the  dentine  from  which  a  narrow  space  often 
appears  to  separate  them  owing  to  removal  of  uncalcified  matrix 
by  the  reagent   and   across   which   the  Jibres  of  Tomes  run  from 


46  DEVELOPING     TOOTH. 

the  odontoblasts  into  the  dentine  tubules.  The  papillary  tissue 
is  embryonic  connective  tissue  in  which  capillaries  and  nerves 
ramify.  After  the  crown  of  the  tooth  has  been  completed,  the 
fang  is  produced  in   a   like  manner,   but   without  enamel. 

The  space  required  for  the  enlarging  tooth  is  provided  by 
the  growth  of  the  jaw  and  the  concurrent  removal  of  osseous 
material  on  the  inner  aspect  of  the  alveolar  cavity  by  the 
osteoclasts,  a  number  of  which  are  observable  upon  the  alveolar 
tissue  around. 

The  dental  germ  for  the  permanent  or  secondary  tooth  occurs 
as  a  small  lateral  offset  from  the  strand  of  epithelial  material 
(gubernaculum),  which  still  connects  the  enamel  organ  with 
the  epithelium  of  the  gum. 


CHAPTER    VI.  11 

MUSCLE. 

Non-striped  muscle.  Small  intestine  of  cat.  (p.  11,  s.  22, 
m.  B.)  Tease  a  piece  of  the  muscular  coat  so  as  to  dis- 
sociate the  fibres  ;  (//)  find  isolated  fibres,  fusiform  in  shape, 
with  elongated  nuclei. 

Stomach  of  a  cat.  T.S.  (p.  3,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find 
the  muscular  coat  composed  of  two  layers  of  muscular  fibre,  one 
cut  longitudinally  the  other  transversely.  In  the  latter  the 
fibres  appear  as  polygonal  areas  united  to  each  other  by  a 
cementing  substance,  across  which  fine  bridge-like  connections 
may  sometimes    be   observed. 

The  frog's  bladder  (Fig.  4)  should  be  prepared  as  follows,  to  show 
this  kind  of  fibre.  Excise  the  organ,  slit  it  open,  and  spread  it,  peritoneal 
surface  downwards,  upon  a  slide  ;  scrape  the  epithelium  off  by  stroking 
it  with  the  pad  of  the  finger,  the  tissue  will  be  partly  dried  and 
will  adhere  to  the  slide  as  a  nearly  transparent  film.  Treat  with  a 
few  drops  of  absolute  alcohol,  which  will  turn  it  opaque,  cover  it 
with  a  pool  of  hsematoxyline,  and  observe  the  staining  taking  place ; 
as  soon  as  it  is  deep  enough  rinse  with  water,  dehydrate  with  alcohol, 
to  which  a  small  quantity  of  eosine  should  be  added  as  a  ground 
stain,  complete  the  dehydration  and  mount  in  balsam. 

(L)  A  network  of  muscular  bands  of  various  sizes  forms  the 

substance    of    the    vesicular  wall,  across    the    meshes   of   which 

(H)  isolated    fibres,  with   well-marked    nuclei,  are  encountered. 

Three-branched  fibres  are  occasionally  met  with.     Blood  vessels 

containing     distorted     corpuscles     (nucleated)     also     unremoved 

surface  epithelial  cells  are  to  be  recognised. 

Cardiac  muscle.  Teased  of  sheep,  (p.  11.)  Dissociate  some 
of   the    tissue   in   a   drop    of    picrocarmine,    the    tissue    cannot 


48  MUSCLE. 

be  teased  into  separate  fibres  for  any  length,  as  it  tends 
to  break  off  short.  (L)  Note  the  branching  nature  of 
the  fibres.  (H)  The  well-marked  cross  and  faint  longitudinal 
striation,  the  nucleus  in  the  substance  of  the  fibre,  and  the 
abrupt  transverse  cleavage  into  short  segments.  These  segments 
are  the   tissue   units. 

Cardiac  muscle.  L.S.  Man  or  sheep,  (p.  2  {d),  s.  22  &  24,  c.  P., 
m.  B.)  (II)  Note  the  transverse  cement  lines,  stained,  which 
define  the  segments.  Each  segment  possesses  a  nucleus.  In  thin 
sections  the  latter  may  often  be  cut  away  and  a  segment  may 
thus  appear  to  be   devoid    of  a  nucleus. 

The  same.  T.S.  Observe  the  oval  and  often  irregular 
outlines  and  difference  in  size,  the  central  nucleus  and  the 
separation  of  the  substance  of  the  fibre  into  sarco^lasm  and 
muscle  fibrils   (forming   sarcostyles,   bundles   of  fibrils). 

Cardiac  muscle.  Dog.  L.S.  Coloured  injection  (p.  2  (rf), 
17  or  22,  c.  P.,  m.  B.)  (L)  and  (//).  There  is  a  rich  dis- 
tribution of  capillaries  parallel  to  the  fibres  with  intercom- 
munications. The  arteries  and  veins  give  off  their  capillary 
brandies   in   fan-shaped    clusters. 

12  Striped  Muscle.  Fresh  muscle.  Snip  a  small  piece  from  the 
sartorius  or  gastrocnemius  of  the  frog,  tease  it  in  normal  saline,  place 
a  camel's  hair  across  the  fibres,  apply  the  cover-glass  and  press  it 
down  slightly  to  produce  local  crushing  of  the  fibres,  then  remove  the 
hair. 

(L)  The  fibres  are  cylindrical  and  marked  with  shadowy  cross 
lines,  transverse  striation.s,  hence  called  striped  muscle.  Each 
fibre  is  enclosed  in  a  structureless  sheath,  the  sarcolemma, 
recognisable  where  the  substance  of  the  fibre  is  crushed  and 
retracted,  or  where  the  membrane  forms  a  blister-like  pro- 
jection.     Nuclei  are  difficult  to  recognise. 


crab's   muscle.  49 

Action  of  acetic  acid.  Irrigate  with  a  drop  of  the  reagent, 
the  fibre  becomes  transparent  and  the  nuclei  are  seen  distinctly 
inside  it. 

Crab's  muscle,  fully  extended,  (p.)  Rutherford's  method.  Crab's 
legs  are  fastened  to  pieces  of  wood  in  the  positions  of  flexion  and  of 
extension.  The  flat  sides  of  the  carapaces  of  the  proximal  segments 
are  removed.  Treat  as  follows  : — 1.  Fix  24  hours  in  formol  (1  to  9  water). 
2.  24  hours  to  some  days  in  methylated  spirit.  Tease  a  piece  of  the 
muscle  finely  in  a  longitudinal  direction,  treat  it  for  two  minutes  with 
glacial  ascetic  acid,  wash  with  water,  stain  in  strong  aqueous  eosin  two 
minutes,  wash  and  mount  in  glycerin. 

The  broad  dim  stripe  is  stained  of  a  deep  red,1  and  consists 
of  a  number  of  rodlike  bodies  (sarcous  elements)  with  a  slight 
central  swelling  (position  of  Hensen's  stripe).  The  light  stripe 
exhibits  the  narrow  dim  stripe  or  Dobie's  line  (Krause's  mem- 
brane) running  across  it ;  to  each  side  of  this  find  egg-shaped 
granules,  FlogeVs  elements  (Englemann's  accessory  disc).  The 
distinctness  of  the  latter  will  depend  upon  the  degree  to  which 
the  muscle  has  been  stretched. 

Crab's  muscle  contracted,  (p.)  In  the  same  manner  as  the 
above.  This  shows  only  a  system  of  dim  stripes  closer  together 
than  and  intervening  light  stripes  without  any  appearance  of 
the  narrow  Dobie's  line.  The  clear  stripe  of  the  uncontracted 
state  has  in  reality  disappeared  and  the  present  dim  stripe 
occupies  its  position.  What  now  appears  as  the  light  stripe 
occupies  the  place  of  Henson's  stripe.  The  stripes  are  thus 
reversed. 

To  see  the  broadening  of  contracting  fibres  the  living  structure 
must  be  examined2,  such  as  the  leg  muscle  of  dytiscus  marginalis. 
or  hydrophilus  pisceus,  in  salt  solution  or  white  of  egg. 

'Rutherford  used  the  term  "chromatin"  to  designate  the  stained  portion.. 
2  See  tongue  of  frog,  pg.  63. 


50  MUSCLE. 

Isolated  muscular  fibres  of  kitten,  (p.  34,  Morpurgo's  method.) 
A  dissociated  fragment  of  a  muscle  is  given  to  you  upon  a 
slide,  spread  a  little  glycerine  upon  it,  and  carefully  lay  the 
fibres  apart  with  needles,  keep  the  fibres  quite  straight. 
Examine  (LJ  and  further  dissociate  until  complete  isolation 
of  a  few  fibres  has  been  accomplished.  The  entire  length  of 
the  fibres  will  be  seen,  cover  and  examine  (H)  and  sketch 
the  ends. 

Muscle  for  muscle  spindles.  T.S.  Sartorius  of  child  or  small 
animal,  (p.  3,  s.  22  &  24  c.  P.,  m.  B.)  (LJ  In  the  Inter- 
fascicular tisue  find  small  blood  vessels  surrounded  by  what 
appears  as  an  unusual  quantity  of  connective  tissue.  (H)  If 
a  muscle  spindle  has  been  hit  upon,  the  T.S.  of  a  few  muscular 
fibres  will  be  recognised  accompanied  by  small  blood  vessels. 
These  are  the  presumed  end  organs  for  the  muscular  sense. 

Tongue  of  dog  or  cat.  T.S.  Coloured  injection.  Recognise 
muscular  fibres  cut  in  various  directions,  note  the  longitudinally 
seen  fibres  of  the  transversus  linguse,  attached  to  the  fibrous 
tissue  of  the  central  "Raphe"  of  the  tongue  on  the  one  hand, 
and  on  the  other  to  the  submucous  connective  tissue.  (H) 
Fibres  in  transverse  section  show  nuclei  beneath  the  sarco- 
lemma  and  the  sarcoplasmic  network  between  the  fibrillse. 
This  feature,  not  equally  distinct  in  all  fibres,  is  more 
evident  in  young  muscle.  (L)  The  blood  vessels  are  filled 
with  a  blue  coloured  mass,  the  capillaries  run  parallel  to 
the  muscular  fibres,  and  are  united  at  intervals  by  cross 
branches. 


CHAPTER    VII.  13 

NERVE. 

Medullated  Nerve.  Fresh,1  Tease  in  normal  saline  a  piece  of 
the  sciatic  or  the  dorsal  cutaneous  nerve  of  a  frog,  cover  and  recognise. 

(Z)  the  cylindrical  fibres  colourless  and  transparent.  (//) 
Single  out  a  fibre,  note  its  smooth  appearance  and  double 
outline.  Find  a  node  of  Banvier,  a  constricted  interruption  of 
the  medullary  sheath,  which  occurs  at  long  intervals  (1  mm)  on 
the  fibre,  very  soon  the  sheath  begins  to  lose  its  smooth  appear- 
ance, through  the  swelling  of  the  myeline,  and  exhibits 
corrugations.  Just  at  the  commencement  of  this  change,  at 
short  intervals,  the  myeline  exhibits  slight  inflections,  or 
actual  oblique  slits,  indicating  a  subdivision  into  shorter 
segments.  These  are  the  "  incisures."  The  medullary  sheath 
appears  to  be  made  up  of  short  segments  (Schmidt-Lantermann), 
the  ends  of  which  fit  into  each  other.  The  semi-fluid  myeline 
in  the  living  condition,  does  not  show  these  planes  of  cleavage, 
and  hardening  reagents  do  not  always  bring  them  into  view, 
or   with   the   same   appearance. 

Osmic  nerve.  Tease  in  glycerin  some  nerve  which  has  been  in 
^p-c-  osmic  acid  solution  for  24  hours,  well  washed  with  water,  and 
stained  in  picrocarmine  for  several  daj-s,  and  then  placed  in  glycerin. 

[L]  The  myeline  of  the  fibres  is  stained  black,  and  there 
is  connective  tissue  between  them  stained  pink.  [H)  The 
nerve  fibres  are  not  all  of  the  same  size,  and  the  smaller 
ones,  owing  to  the  thinness  of  their  medullary  coat,  appear 
only  faintly  stained.     Find    a    node   in    an   isolated    fibre,   note 

1  Nerves  in  a  living  condition.      See  tongue  of  frog,  pg.     ::,. 


Q'J,  NERVE. 

the  slightly  bulbous  enlargement  which  precedes  the  constric- 
tion of  each  end.  Find  a  second  node  along  the  same 
fibre,  observe  also  that  the  medullary  sheath  now  shows  the 
incisures  distinctly,  owing  to  slight  shrinkage  of  its  substance. 
The  nuclei  stained  pink  occupy  depressions  in  this  sheath, 
and  are  best  seen  in  profile  view.  There  is  one  nucleus  to 
each  internode  (Ranvier).  The  axis  cylinder  may  project  from 
the  broken  end  of  a  fibre. 

Xerve  treated  tvith  nitrate  of  silver.  Fresh  nerve  is  placed  in 
lP-cAgNOs  for  20  minutes,  then  transferred  to  30  p-c-  alcohol  in 
sunlight   until   darkened.      Tease   a   piece   of   the  nerve   in   glycerin. 

(//)  Find  Ranvier' s  crosses,  nodes  where  the  brown  stain- 
ing exhibits  the  axis  cylinder  and  nodal  cement  substance 
coloured,  roughly  cross-shaped.  The  staining  may  extend  along 
the  axis  cylinder,  and  then  is  disposed  as  transverse  markings 
(Frommann's  striae).  This  disposal  of  the  stain  indicates 
the  existence,  around  the  axis  cylinder,  of  material  corres- 
ponding to  the  cement  at  the  node.  Incisural  outlines  may 
be  indicated. 

Large  nerve.  T.S.  Sciatic  or  ulnar,  man.  (p.  11,  s.  22,  c.  P.,  m.  B.) 
(L)  The  large  rounded  areas  are  bundles  of  nerve  fibres.  The 
perineurium  around  each  bounds  them  with  a  sharp  out- 
line ;  between  and  embracing  them  is  loose  connective  tissue, 
epineurium.  (II)  The  perineurium  is  a  lamellated  structure 
consisting  of  several  layers,  between  which  there  are  nuclei  and 
lymph  spaces.  Internally  to  this  sheath  are  the  nerve  fibres 
mostly  seen  in  transverse  section.  Each  nerve  exhibits  a  stained 
axis  cylinder,  around  which  is  the  medullary  sheath. 

This  sheath  varies  in  its  appearance  with  the  method  of 
preparation.  If  long  hardened  in  Midler's  fluid  it  is  more 
homogeneous    and    shows,    here   and    there,   concentric   outlines 


NON-MEDULLATED    NERVE.  53 

due  to  the  incisures.      If    hardened  in    Bulpho-picric    acid    (15) 
it  presents  a  radiating  structure. 

The  neurilemma  forms  a  sharp    boundary  to  the  fibre.     The 
endoneurium  supplies  connective  tissue  septa  in  the   interfasci 
cular  spaces  of  the  nerve  bundle,  in  it  are  found   blood   vessels 
and  lymph  spaces. 

Oxmic  nerve.  T.S.  48  hours  in  1  p-c-  osmic  acid,  48  hours  absolute 
alcohol,     (c.  P.,  m.  B.) 

(Z)  General  features  the  same  as  in  the  last  preparation. 
(//)  The  medullary  substance  being  blackened,  the  nerve  fibres 
appear  as  black  rings.  Observe  the  great  difference  in  the 
sizes  of  the  nerve  fibres  in  the  same  bundle.  The  largest  are 
chiefly  motor  (Gaskell),  some  are  intermediate  in  size  and  others 
very  small  (visceral  ?). 

Nerve.  L.S.  (p.  11,  s.  22,  c.  P.,  m.  B.)  (L)  The  nerve  fibres  are 
wavy  in  their  arrangement  inside  the  perineurium.  (//)  Single 
out  a  nerve  fibre  which  can  be  followed  for  some  distance.  The 
axis  cylinder  is  stained,  so  is  also  to  a  lesser  degree  the  neuri- 
lemma. In  the  space  occupied  by  the  medullary  sheath  funnel- 
shaped  structures  are  visible  (Golgi's  funnels).  These  correspond 
to  the  incisures.     The  nodes  can  also  be  recognised. 

Non-mecLullated  Nerve.  Splenic  Nerve.  T.S.  (p.  12, 
s.  22  &  24,  c.  P.,  m.  B.)  (L)  The  perineurium  forms  a  well- 
marked  envelope  to  the  bundle  of  nerve  fibres.  The  latter 
exhibit  little  noticeable  structure  beyond  small  ill-defined 
transverse  sections,  in  which  occasional  nuclei  are  seen. 
Scanty  interfascicular  connective  tissue  septa  divide  the  mass, 
in  these  capillaries  are  present. 

L.S.  of  the  same  nerve  exhibits  wavy  fibres  crowded  together. 
Each  nerve  fibre  is  nucleated  and  practically  corresponds  fco  an 
axis  cylinder. 


54  NERVE. 

^4        Nerve  Cells-     Multipolar  nerve  cells  of  the  grey  matter  of 

the  Spinal  Cord  of  a  large  mammal. 

Remove  a  small  piece  of  the  anterior  horn  of  the  grey  matter  of  the 
fresh  cord,  and  compress  it  to  a  thin  film  between  two  cover-glasses,  slip 
them  off  each  other  and  stain  the  films  with  methylene  blue  for  one  minute, 
quickly  rinse  them  in  water  and  dry  thoroughly  in  air  over  the  bunsen 
flame,  mount  by  inverting  them  when  quite  dry  upon  a  drop  of  balsam 
on  a  slide. 

(//)  Find  large  cells  stained  blue,  they  are  nucleated  and  their 
protoplasm  shows  the  chromatic  patches  deeply  stained,  and  the 
fibrillar  components  of  the  larger  processes  continued  in 
a  radiating  manner  into  the  protoplasm.  A  process,  the  axon, 
which  does  not  branch  is  often  recognisable.  Other  processes 
form  rich  arborescent  ramifications.  Note  the  numerous  nuclei 
which  belong  to  the  neuroglia.  Branching  blood  vessels  are 
also  met  with. 


CHAPTER    VIII. 
CIRCULATORY    SYSTEM. 

Small  artery  and  vein.  T.S.  from  pancreas.  (p.  4, 
s.  22  &  24,  c  P.,  m.  B  )  (L)  The  artery  is  recognised  by  the 
greater  thickness  of  its  walls.  Three  tunics  are  recognisable. 
The  inner  (intima),  just  discernable  under  this  power,  shows 
the  wavy  internal  elastic  lamina,  on  the  inner  side  of  which 
nuclei  may  be  perceived ;  outside  this  is  the  thicker  middle 
coat  (media),  consisting  of  non-striped  muscular  fibres  ; 
and  externally  is  the  outer  coat  (adventitia)  of  connective 
tissue.  (H)  The  inner  coat  has  an  inner  lining  of  endothelium, 
chiefly  recognisable  by  the  nuclei  of  its  cells,  beneath  this 
lies  the  internal  elastic  membrane  (fenestrated  membrane  of 
Henle),  and  between  the  two — hardly  recognisable,  owing  to 
its  thinness — is  the  sub-endoihelial  connective  tissue.  The 
media  or  muscular  coat  in  an  artery  of  this  size  consists 
entirely  of  muscle.  The  adventitia  generally  shows  elastic 
fibres  near  the  muscular  coat :  not  marked  in  very  small 
arteries.  The  characteristic  feature  of  the  small  artery  is  its 
purely   muscular  coat. 

The  vein  differs  from  the  artery  in  having  thinner  walls, 
the  same  structures  occur  in  both.  The  muscular  coat, 
however,  frequently  contains    fine    elastic  tissue. 

Distended  blood  vessels.  T.S.  artery  and  vein  ligatured  in  a  dis- 
tended condition  (p.  3,  s.  22  &  24,  c.  P.,  m.  B.). 

(L)  The  artery  shows  a  perfectly  smooth  and  unfolded 
internal  elastic  lamina,  and  its  walls  appear  proportionately 
thinner.       The  same  is  observable  of  the  vien. 


56  CIRCULATORY    SYSTEM. 

Endothelial  outlines  in  blood  vessels.  Intestine  of  a  small 
mammal,  injected  with  AgN03  (27)  and  p.  in  methylated  spirit. 
Spread  the  opened  intestine  peritoneal  surface  downwards  on  the  slide, 
and  scrape  away  the  mucous  coat  with  the  back  of  a  scalpel,  clear 
with  clove  oil,   mount  in    balsam. 

(L)  A  branching  system  of  blood  vessels  in  which  the  arteries 
are  smaller  than  the  veins,  their  branches  being  united  by  capil- 
laries. (H)  Endothelial  outlines  of  the  arteries  are  more  fusifom 
than  those  of  the  veins.  Trace  the  passage  of  the  arteries 
into  the  capillaries.  Observe  that  the  cell  territories  in  the 
latter  are  larger  and  somewhat  jagged.  Around  the  arteries 
perivascular  lymphatics  are  sometimes  observed.  Their  endo- 
thelial ceils  have  sinuous  outlines. 

Aorta.  T.S.  (p.  3,  s.  19,  c.  P.,  m.  B.)  (L)  The  middle  coat  is 
the  predominant  structure,  and  consists  largely  of  eristic  tissue 
in  circularly  distributed  networks  interconnected  in  all  directions. 
In  its  meshes  lie  muscular  fibres.  On  the  inner  aspect  is 
the  thin  intima,  and  externally  is  the  adventitia,  with  the 
same  structure  as  before.  The  abundant  elastic  tissue  in 
the  middle  coat  is  the  characteristic  feature  in  the  large  artery. 

Sinus  of  valsalva.  L.S.  Man  (p.  11,  c.  G.,  s.  19,  m.  F.).  (L) 
Recognise  the  aorta,  trace  it  to  its  junction  with  the  base  of  the 
ventricle,  a  portion  of  which  is  included  in  the  section,  the 
junction  is  effected  by  the  intervening  connective  tissue  of  the 
Tendo  cordis,  stained  pink.  On  one  side  of  this  junction  there  is 
loose  connective  tissue  which  leads  into  the  substance  of  the  semi- 
lunar valve,  which  consists  of  open  connective  tissue,  more  com- 
pact on  its  ventricular  aspect,  and  covered  on  both  sides  with 
endothelium.  The  recess  between  the  aorta  and  the  valve  is  the 
sinus.  Note  that  the  elastic  tissue  of  the  wall  of  the  aorta  thins 
down,  and  is  replaced  by  pink-stained  connective  tissue  where 
it  forms  the  wall  of  the  sinus.      (//)   Having  noted  the  thinning 


VENTRICLE. — BLOOD.  5  i 

of  the  elastic  wall  of  the  aorta,  and  the  fcendo  cordis,  recognise 
the  cardiac  muscle  and  its  mode  of  attachment  to  the  latter 
structure,  examine  the  substance  of  the  valve,  observe  its 
covering  of  endothelium. 

Should  the  section  pass  through  the  corpus  arantii  its  structure 
will   be  recognised   as   white   fibro-cartilage. 

Ventricle  of  Sheep.     V.S.     (p.  2  {d),  s.  22  &  24,  c.  P.,  m.  B.; 

(L)  The  myocardium  consists  of  muscle,  sub-divided  into  fasciculi, 
separated  by  thin  connective  tissue  septa,  is  covered  on  its  inner 
aspect  by  the  thin  endocardium  which  follows  all  the  irregu- 
larities of  the  surface  and  upon  the  external  aspect  by  the 
pericardium.     The  latter   often   contains   much    fat. 

(H)  The  endocardium  loose  connective  tissue,  in  which  are 
a  few  fibres  of  non-striped  muscle,  exhibits  a  number  of  elon- 
gated oval  masses,  the  fibres  of  Purkinje.  These  consist  of  large 
polygonal  cells,  containing  one  or  two  distinct  nuclei,  surrounded 
by  undifferentiated  protoplasm.  The  periphery  of  each  cell 
is  fibrillated  parallel  to  its  surface.  Trace  them  into  the 
myocardium  and  note  their  transition  into  cardiac  muscle. 
These  fibres  occur  in  ruminants  (Purkinje),   not  in  man. 

BLOOD.  15 

Amphibian  and  human  blood.  Study  these  successively 
in  the  same  manner.     Obtain  them  as  follows. 

Amphibian  Blood.  Frog.  Expose  the  heart  of  a  pithed  frog 
freely,  draw  it  over  the  edge  of  a  watch  glass  with  forceps  and  snip 
into  the  ventricle.  Collect  the  outflowing  blood  without  admixture 
of  moisture  from  the  skin  and  touch  the  blood  with  covers  or  hold 
covers  so  that  the  blood  may  flow  upon  them.  Newt.  Dry  the  tail, 
snip  off  the  end.  Apply  covers  to  the  blood  that  collects  on  the 
stump  without  touching  the  skin. 

Human.  Congest  the  end  of  your  finger  by  winding  a  cloth  firmly 
round  it  from  base  to  point.  With  a  clean  Glover's  needle  prick  the 
skin  near  the  root  of  the  nail  until  the   blood  wells  out    freely,  touch 


58  CIRCULATORY    SYSTEM. 

the  drop  with  covers.  The  covers  are  then  placed  upon  slides  or 
otherwise  dealt  with  as  in  the  sequel.  There  should  be  enough  blood 
between  the  glasses  to  form  a  continuous  film  devoid  of  air  bubbles. 

Freshly  shed  blood.  To  retard  changes  draw  a  line  of  olive  oil 
round  the  edge  of  the  cover  and  examine  at  once. 

(//)  The  formed  elements,  blood  corpuscles  or  cells,  floating 
in  a  transparent  colourless  fluid  the  blood  plasma.  Recognise 
the  numerous  red  and  scanty  white  cells.  The  latter  appear 
bluish  by  contrast,  and  when  slightly  out  of  focus  more 
luminous   than   the   red. 

Red  blood  corpuscles.  Examine  them  (H)  carefully  as  to 
shape,  relative  and  actual  size,  colour,  contents,  and  note  if 
there  be  any  varieties.  The  normal  amphibian  red  cell  does 
not  exhibit  its  nucleus.  The  nucleus  becomes  prominent  as 
changes  supervene,  and  is  readily  extruded  from  the  cell.  It 
is  oval,  the  karyoplasmic  network  is  distinct  and  colourless. 
Do   not   confound  escaped   nuclei   with    white   cells. 

The  human  red  cells  tend  shortly  to  cohere  by  their  broad 
surfaces,  similating  piles  of  coins  (rouleaux),  probably  due  to 
altered  surface  tension.  The  form  of  the  individual  cell, 
a  biconcave  disc,  is  recognised  as  it  revolves  and  is  seen  on 
edge.  On  the  flat  their  lenticular  shape,  i.e.,  the  marginal 
biconvexity  and  central  biconcavity  causes  a  faint  concentric 
shadow  which  moves  from  edge  to  centre,  and  inversely  with 
changes  of  focus.  Not  infrequently  even  at  first  some  of  the 
cells  are  crenated,  i.e.,  have  prickly  outlines. 

In  the  course  of  a  few  minutes  clotting  of  the  blood  may 
occur,  when  very  fine  filaments  of  fibrin  will  be  recognisable 
traversing  the  plasma  (now  serum).  Granules  are  often  to 
be   noted   at   the   points   of  radiation   of   the   filaments. 


REAGENTS  AND  RED  CELLS. LEUCOCYTES.  59 

Effects  of  reagents.     Applied  by  irrigation. 

Water.  Cells  lose  their  distinctness  and  the  colouring 
matter  is  dissolved.  The  outlines  of  single  cells  are  still 
recognisable  where  they  are  few  in  number.  Note  any  change 
in  their  shape  and  size. 

Si/rup.  20 pc-  solution  Cane  sugar.  Note  the  result  of 
exosmosis  on  their  shape. 

Tannic  acid  strong  solution  and  Boracic  acid  2 pc-  solution. 
Compare  their  effect  upon  the  pigment,  which  is  disseminated 
as  granules  within  or  without  the  cells. 

Salicylic  acid.  Half  saturated  solution  in  alcohol.  Compare 
with  the  actions  of  tannin  and  of  boracic  acid. 

Acetic  Acid.  Watch  the  earliest  effect  of  the  reagent  and 
compare  its  effect  on  amphibian  and  human  blood  and  on  the 
nucleus  in  the  former. 

Leucocytes,  white   or  colourless  blood  corpuscles. 

Fresh  blood  shielded  with  oil.  Human  blood  should  be  received  on 
a  warmed  slide,  covered,  and  be  placed  on  the  hot  stage. 

Search  for  three  varieties : — (1)  Large  finely  granular. 
(2)  Large  coarsely  granular.  (3)  Small  round  (Lymphocytes). 
Examine  minutely  their  cytoplasm,  the  nucleus  is  not  clearly 
discernible. 

Amoeboid  movements.  Find  cells  in  active  movement ;  note 
the  variety  to  which  they  belong.  The  resting  spherical  form 
is  lost,  the  cell  becomes  irregularly  extended  by  a  flowing 
motion,  with  resultant  changes  of  shape  and  position.  Sketch 
the  outline  every  30  seconds. 


60  CIRCULATORY    SYSTEM. 

Effect  of  acetic  acid.  The  red  cells  are  cleared  up  and  the 
white  ones  come  prominently  into  view.  The  latter  are 
rendered  transparent  and  their  nuclei  distinctly  visible.  Note 
the  shapes  of   the  nuclei. 

^g  Ppeparations    of  fixed    blood.      Dried    blood  film.      A    cover    is 

moistened  with  blood,  a  second  cover  is  laid  upon  it,  after  a  minute 
the  two  are  slid  apart  and  are  rapidly  dried  in  air.  Invert  upon 
a  slide  and  fasten  down  with  a  piece  of  gummed  label  having  an 
aperture  cut   in  the   centre. 

The  red  cells  are  perfectly  preserved  and  visible  but  the 
leucocytes  are  more  difficult  of  recognition. 

Stained  films.  A  successful  result  depends  upon  the  thinness 
of  the  film. 

1.  Make  several  blood  films  by  rapidly  drawing  the  edge 
of  a  square  cover  moistened  with  blood  across  the  surface  of 
other   covers. 

2.  Dry   them   quickly   by   waving   in   the   air. 

3.  When  perfectly  dry  pass  the  covers,  film  uppermost,  three 
times   through  the   flame   of   the   Bunsen   burner. 

4.  Stain  them  for  10  seconds  in  a  quarter  saturated  alco- 
holic solution  of  eosin.  The  stain  is  applied  by  spreading  a 
drop  of  the  fluid  at  one  stroke  over  the  film  with  the  flat  of 
the   rod   of   the   reagent  bottle. 

5.  Rinse  in   successive   drops  of  water  until  the  stain  ce 
to   discharge. 

6.  Stain  in  the  same  manner  for  30  seconds  in  Luffler's 
in^thelyne    blue. 

7.  Rinse  as   before  in    water. 

8.  Blot  off  the  moisture  by  placing  the  cover  between  the 
folds  of  a  piece  of  filter  paper. 


AMJEKOID     LEUCOCYTES.       FIBRIN.  61 

9.  Dry  thoroughly  in  the  air  with  final  wanning  over  fche 
burner. 

10.   Invert  upon  a  drop  of   balsam   upon  a  slide. 

The  red  cells  are  stained  red  and  their  nuclei,  when 
present,  blue,  the  leucocytes  have  their  nuclei  similarly 
coloured,  and  the  eosinophil  granules  are  red. 

Leucocytes  of  the  Newt  fixed  in  amasboid  extension. 
A  drop  of  newt's  blood  on  a  cover  is  inverted  on  a  drop  of  normal  saline 
and  is  irrigated  with  the  same  to  remove  as  much  of  the  plasma  as 
possible.  The  preparation  is  then  left  to  itself  for  ten  minutes  to  give 
the  leucocytes,  which  have  adhered  to  the  glass,  time  to  be  in  active 
movement.  The  cells  are  next  fixed  (instantaneously)  by  heat.  Play 
a  jet  of  steam  on  the  cover  of  the  preparation,  which  is  held  for  two 
seconds  close  to  the  point  of  issue,  from  a  tube  fitted  to  a  flask  or 
large  test-tube  in  which  water  is  kept  boiling  briskly.  Irrigate  with 
70 pc-  alcohol,  followed  by  dilute  hematoxylin  for  four  minutes.  Wash 
away  the  latter  with  70p-c-  alcohol,  then  absolute  alcohol  tinged  with 
eosin,  followed   by   cedar  oil  and,  lastly,   balsam.     (After   Schafer. ) 

(//)  Find  the  cells  and  observe  the  varieties  of  shape 
assumed  by  the  cytoplasm  and  nuclei,  and  the  distribution  of 
the  eosinophil,   i.e.,  eosin  stained  granules  in  the  former. 

Blood  in  Hayem's  fluid. 1  Mount  a  preparation  made  as  follows  : 
Fresh  blood  is  dropped  into  a  considerable  volume  of  the  fluid  with 
which  it  must  be  thoroughly  mixed  by  gentle  agitation.  After  standing 
from  four  to  five  hours  the  supernatant  fluid  is  decanted  off  and  the 
blood  washed  with  three  changes  of  water  in  the  same  manner.  Small 
quantities  are  stained  with  picrocarmine  (19)  or  hiemalum  (22)  followed 
by  eosin  and  mounted  in  glycerin  or   (Stirling)   glycerin  jelly. 

Fibrin.  A  thick  film  of  blood  is  covered  and  allowed  to  stand 
ten  minutes.  Raise  the  cover,  rinse  the  adherent  film  of  fibrin  free  of 
colour  with  water,  then  invert  on  a  drop  of  Spiller's  purple  on  a  slide 
until  stained  (H)  one  minute,  wash  in  water,  dry  between  folds  of 
blotting  paper,  mount  in  balsam. 

hayem's  fluid.  Sodium  chloride  1  g,  sodium  sulphate  5g,  corrosive  sublimate 
0-5  g,  distilled  water  200  cc. 


62  CIRCULATORY    SYSTEM. 

(H)  The  clot  will  show  delicate  filaments  forming  a  con- 
fused network  of  radiating  and  intercrossing  threads  often  of 
great  fineness.  Clusters  of  granules  occur  frequently  at  the 
points  of  radiation.     Blood  platelets? 

Haemoglobin  crystals.  Rat  or  guinea-pig.  Mix  a  drop  of  blood 
on  a  slide  with  the  same  quantity  of  water,  cover  and  watch  for  the 
appearance  of  crystals,  first  near  the  edge  of  the  cover.  When  well 
developed  remove  the  cover,  dry  thoroughly,  replace  the  cover  and 
cement  it  with  a  thin  edging  of  glycerin  jelly,  covered  when  dry  with 
gold  size. 

Hsemin  crystals.  To  a  drop  of  your  own  blood  on  a  slide  add 
a  small  grain  of  chloride  of  sodium,  nearly  dry  it,  cover  and  add  some 
glacial  acetic  acid  and  heat  over  the  bunsen  flame  until  bubbles  are 
freely  given  off.  Repeat  the  heating  if  the  crystals  do  not  appear.  Diy 
the   preparation   completely,  add  balsam,  and  cover. 

Examine  (II)  for  crystals,  which  will  gradually  form,  appear- 
ing at  first  as  nearly  black,  fine,  short  needles,  often  in 
clusters. 

yi        The  Circulation.    May  be  studied  in  any  of  the  following  : — 

Web  of  the  frog's  foot.  Use  a  piece  of  cotton  cloth  six  inches 
square  with  a  hole  in  the  centre.  Wet  the  cloth,  draw  one  hind  leg 
through  the  hole  and  wrap  the  remainder  round  the  frog  so  as  to  form 
a  sack,  secure  the  mouth  with  string,  and  place  the  frog  upon  the 
.support.  Secure  with  ligatures  around  the  support  and  frog,  one  round 
the  body  and  one  encircling  the  knees.  The  frog  will  remain  quiescent 
if  it  is  kept  moist  and  is  not  too  tightly  restrained  by  the  ligatures.  To 
the  longest  and  one  of  the  neighbouring  toes  of  the  foot  attach  soft  threads, 
lay  the  frog  ventrally  on  the  support  and  extend  the  web  over  the  triangular 
gap  by  securing  the  threads  in  the  slits  cut  for  the  purpose.  Cover  the 
extended  portion  of  the  web  with  a  triangular  piece  of  cover-glass. 

Frog-'s  mesentery.  In  a  pithed  frog  laid  dorsally  on  the  support 
open  the  abdominal  cavity  by  a  longitudinal  incision  below  the  axilla. 
Carefully  draw  out  a  loop  of  the  small  intestine  and  pin  it  to  the  semi- 
circular cork   rim  on  the  support.     Cover. 


CIRCULATION     IN     THE     PROG's     Fool.  63 

Tongue  of  frog.1  Evert  the  tongue  of  the  pithed  animal  out  of 
its  mouth  and  spread  it  fanwise,  avoiding  over  extension,  fixing  in 
position  to  the  cork  with  short  pins.     Cover. 


Fig.  14.     Frog  support  for  studying  the  circulation  in  A  the  tongue,  B  the  mesentery, 

and  C  the  web  of  the  foot. 

The  foregoing  offer  different  aspects  of  the  subject  for  study.  The 
web  being  covered  by  skin,  the  pigment  and  guanin  cells,  sometimes 
very  numerous,  may  interfere  considerably  with  a  clear  view  of  the 
vascular  structures  beneath.  Capillaries  are  numerous.  The  mesentery 
being  very  thin  shows  the  larger  vessels  well,  but  there  are  few  capillaries. 
The  tongue  exhibits  large  tortuous  (Lingual)  arteries  in  which  the  expan- 
sion at  the  ventricular  systole  is  particularly  well  seen. 

Circulation  in  the  web.  Search  (L)  for  an  artery,  the  blood 
stream  runs  in  it  from  the  trunk  into  the  branches;  find  a  vein, 
in  it  the  streams  converge  into  the  trunk.  Note  the  relative 
difference  in  the  rate  of  flow  and  in  the  diameter  of  the  two 
vessels.  Trace  the  blood  stream  from  the  artery  into  the  vein 
through  the  capillaries.  (H)  Study  the  flow  in  the  latter.  In 
these  the  blood  cells  are  individually  visible,  the  red  ones  bending 
to  the  curves  around  which  they  travel ;  occasionally  a  red 
cell  is  caught  on  the  edge  of  bifurcation  of  a  vessel  it  then 
becomes   flexed    and    responds    to   each    systole,    recovering   its 

1  In  this  organ  living  muscle  and  nerves  can  be  observed.  When  thus 
extended  striped  muscular  fibres  may  be  readily  fixed  and  isolated  as  follows : — 
Drop  absolute  alcohol  upon  the  mucosa  until  bleached,  scrape  through  it  in  the 
direction  of  the  bands  of  muscle  until  the  latter  are  exposed,  and  let  the  alcohol 
act  upon  them  until  they  lose  their  elasticity.  Remove  portions  and  immerse  them 
in  absolute  alcohol  for  twenty  minutes.  Separate  by  teasing  and  after  bathing 
with  water  stain  them  with  hematoxylin  and  mount  in  balsam. 

Small  nerve  bundles  accompany  the  blood  vessels,  the  outlines  of  the  fibres  are 
sharply  denned,  and  the  double  contour  of  the  medullary  sheath  and  Ranviers 
nudes  can  be  recognised.  In  the  thin  edges  of  the  preparation  search  for  single 
fibres  of  striped  muscle,  ami  observe  the  occasional  waves  of  contraction. 


G4  CIRCULATORY    SYSTEM. 

shape  on  being  swept  back  into  the  stream.  Capillaries  at 
times  change  their  calibre  and  become  too  narrow  for  the 
passage  of  the  red  cells.  The  leucocytes  are  swept  along  by 
the  general  blood  stream  ;  they  tend,  however,  to  attach 
themselves  to  the  wall  of  the  vessel,  and  can  be  seen  accumu- 
lating upon  the  surface  whenever  the  current  slows  sufficiently. 
In  the  veins  where  the  flow  is  less  rapid  than  in  the  arteries 
the  leucocytes  monopolise  a  peripheral  space  in  which  they  may 
be  seen  to  be  rolled  along  by  the  current  as  they  cling  to 
the    wall   of  the  vessel. 


CHAPTER    IX. 
RESPIRATORY    SYSTEM. 

Trachea.  T.S.  Dog.  (p.  8,  s.  22*»  &  24,  c.  P.,  m.  B.)  (Z)  An 
incomplete  ring  of  cartilage,  the  posterior  ends  of  which 
overlap  and  are  united  by  the  trachealis  muscle.  The  peri- 
chondrium passes  internally  into  the  looser  submucous  tissue, 
in  which  note  mucous  glands.  The  inner  surface  has  a 
well-defined  edge,  which  recognise  (//)  to  consist  of  many 
layers  of  epithelium.  The  surface  cells  are  columnar-ciliated. 
Examine  the  shapes  of  the  cells  in  the  different  layers  and 
the     surface    upon    which     they     rest. 

Large  bronchus  in  the  lung.  Sheep  or  cat.  (p.  8, 
s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  bronchi  cut  across,  in 
bronchial  tracts,  surrounded  by  but  sharply  mapped  off  from 
the  vesicular  lung  substance.  The  thick-walled  bronchus  is 
associated  with  the  pulmonary  artery  and  vein,  which  usually 
lie  on  opposite  sides  of  it  to  form  the  tract,  an  arrangement 
which  becomes  less  regular  peripheralwards.  (II)  The  epithelial 
layer  of  the  mucosa  lining  the  inner  surface  of  the  bronchus 
is  wavy  (sectional  view  of  longitudinal  corrugations),  and  per- 
forated by  the  ducts  of  the  mucous  glands.  Externally  beneath 
the  sharply-defined  surface  of  attachment  of  the  epithelium 
note  the  layer  of  transversely  cut  elastic  fibres,  and  next  a 
continuous  band  of  non-striped  (bronchial)  muscle.  Between 
the  latter  and  the  plates  of  cartilage  are  disposed  the  mucous 
glands,  interspersed  with  variable  quantities  of  adenoid  tissue 
(lymph  cords,  Klein),  and  areolar  tissue.  The  perichondria! 
extensions    form    a    continuous    fibrous    covering    around    the 


66  RESPIRATORY    SYSTEM. 

bronchus.  Recognise  the  large  pulmonary  artery  and  vein 
surrounded  by  loose  peribronchial  connective  tissue,  and  the 
sharp  delimitation  of  the  latter  by  the  vesicular  tissue  of  the 
lung.  Search  for  the  small  bronchial  arteries  scattered  around 
the  bronchus  (which  convey  arterial  blood  to  its  tissues)  and 
the  nerves  with  frequent  ganglia  which  accompany  them. 
Endeavour  to  trace  in  the  surrounding  lung  substance  the 
expansion  of  the  bronchus  into  the  vesicular  tissue  and 
examine  the  latter. 

Silvered  Lung  of  kitten  or  other  young  mammal.  The  freshly 
excised  lung  is  filled  with  0*25  p-c-  solution  AgN03  through  a  funnel 
tied  into  the  trachea,  the  air  driven  out  by  gentle  squeezing,  then 
alternately  emptied  and  refilled  once  with  silver  solution,  and  twice 
with  methylated  spirit.  The  second  charge  of  the  latter  being  made 
to  distend  the  lung  is  retained  by  ligaturing  the  trachea,  the  whole 
being  finally  immersed  in  spirit.  When  rigid,  48  hours,  cut  into  con- 
venient pieces  and  continue  the  hardening  14  or  more  days  in  sunlight 
(c.  G,  s.  19,  m.  F.  or  B.). 

(L)  Recognise  the  bronchi,  find  an  L.S.  bronchiole  at  its 
infundibular  expansion  (//),  note  the  cubical  epithelium  of 
the  bronchiole,  the  absence  of  cartilage  and  thinness  of  its 
walls.  Study  closely  how  the  vesicular  recesses  (air  vesicles), 
multiple  saccular  expansions,  are  connected  with  the  walls  of 
the  infundibular  end  of  the  bronchiole.  The  portions  which 
remain  of  the  latter  may  be  recognised  around  the  vesicular 
orifices  by  their  covering  of  cubical  epithelium.  The  endothelial 
lining  of  the  air  sacs  is  outlined  by  the  action  of  the  silver, 
each  cell  possessing  a  nucleus.  Smaller  cells  stained  brown 
occur  singly  or  in  groups  of  two  or  three  between  the  larger 
clear  ones,  and  may  be  regarded  as  germinal  cells  or  as 
pseudostomata  connected  with  the  perivesicular  lymphatics. 
Distinguish  on  the  outer  surface  of  the  lung  the  pleural 
epithelium  and  connective  tissue. 


F(ETAL     AND     INJECTED     LUNG.  07 

Foetal  lung-.  Man.  (p.  14(6),  s.  22,  c.  P.,  m.  B  .)  (L)  The  jg 
subdivision  of  the  lung  into  lobules  attached  centrally  to  the 
larger  bronchi.  Between  the  former  note  the  loose  interlobular 
connective  tissue  continuous  with  the  pleural  tissue  on  the 
surface  of  the  lung.  In  the  lobules  the  ramifications  of  the 
air  passages  are  discernible.  (H)  The  pleural  and  interlobular 
connective  tissue  exhibit  blood  vessels  and  numerous  lymphatics. 
The  branchings  of  the  incompletely  developed  bronchi  can  be 
synthetically  followed  to  their  terminations,  which  may  not 
however  be  sufficiently  developed  to  present  air  vesicles,  but 
merely  the  blind  terminations  of  the  bronchioles.  Note  the 
large    amount  of  interstitial   connective   tissue. 

Injected  Lung".  Mount  a  section  of  lung  the  blood-vessels 
of  which  have  been  filled  with  a  coloured  gelatin  mass. 
(L)  Find  the  large  pulmonary  vessels  filled  with  the  coloured 
mass  and  (H)  recognise  the  injected  capillaries  which  form 
a  close  network  in  the  walls   of  the  air  vesicles. 


CHAPTER   X. 

ALIMENTARY   CANAL. 

Tongue.  T.S.  Kitten,  (p.  3,  inj.,  s.  22  &  24  c  P.,  m.  B.)  (L) 
The  dorsal  surface  of  the  organ  is  fringed  with  papillcn,  which 
are  absent  elsewhere.  The  superficial  stratified  epithelium  has 
beneath  it  fibrous  tissue,  these  together  constitute  the  "Mucosa," 
the  papilla?  being  formed  by  projections  of  the  two  layers. 
Internally  the  organ  is  muscular  and  is  divided  symmetrically 
into  lateral  halves  by  a  thin  fibrous  septum  (Raphe).  Note  the 
strata  of  the  transverse  muscle  alternating  with  those  of  the 
vertical  muscle.  The  fibres  of  these  are  attached  to  the  septal 
and  mucosal  fibrous  tissue.  The  other  intrinsic  muscles  are 
the  dorsal  and  inferior  Unguals.  Near  the  latter  find  sections 
of  small  blood  vessel  and  nerves.  (//)  Examine  the  conical 
papilla?,  observe  the  fibrous  core  and  the  enveloping  squamous 
epithelium.  Search  the  sub-mucosa  for  mucous  glands,  there  will 
be  none  unless  the  section  is  taken  from  the  hinder  part  of 
the  tongue. 

Tongue.  V.8.  Middle  of  dorsum,  Man.  for  Fungiform  papilla. 
(p.  11,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  and  (H)  The  papilla?  in 
question  are  shaped  like  button  mushrooms,  the  surface  of  the 
fibrous  core  exhibits  small  secondary  papilla?. 

Tongue,  V.S.,  base  of,  Man.  for  circumvallo •/ )e  papillce.  (p.  21, 
b.  22  &  24,  c.  P.,  in.  B).  (L)  Find  a  circumvallate  papilla  and 
study  its  structure  in  conjunction  witli  that  of  the  corresponding 
organ  in  the  next  preparation,  the  details  of  which  are  better 
preserved  as  the  tissue  can  be  fixed  in  a  fresher  condition. 


PAPILLA     POLIATA. PALATE.  69 

Papilla  foliata.  T.S.  of,  in  the  root  of  the  rabbit's  tongue, 
(p.  3,  s.  22  &  24,  c.  P.,  m.  B.)  The  papilla  projects  slightly 
above  the  general  level  of  surface  of  the  tongue  and  consists 
of  a  series  of  vertical  clefts  (sulci)  which  indent  its  surface, 
and  are  lined  by  the  superficial  stratified  epithelium.  In 
this  epithelium,  on  each  side  of  the  sulcus,  the  taste  buds 
are  placed.  Find  the  secreting  portions  of  the  serous  glands 
situated  at  some  depth  in  the  muscular  tissue  of  the  tongue 
and  trace  their  ducts,  which  open  into  the  sulci.  Outside 
the  foliate  area  mucous  glands  similarly  disposed  will  he 
found,  and  are  easily  recognised  by  the  transparent  character 
of  their  epithelium.  (H)  Examine  the  taste  buds.  They 
are  oval  masses  composed  of  fusiform  cells  arranged  after  the 
fashion  of  the  staves  of  a  barrel,  the  axis  of  which  is  set 
vertically  to  the  sulcus.  The  pointed  apex  of  the  bud  cor- 
responds to  an  opening  in  the  surface,  the  gustatory  pore, 
through  which  the  inner  set  of  cells  of  the  bud  project  as  a 
cluster  of  minute  short  bristles.  Outside  the  bases  of  the  buds 
modified  fibrous  papilla?  form  an  access  for  the  nerves  going 
to  the  buds.  Compare  the  cells  in  the  serous  glands  with 
those  in  the  mucous  glands  and  find  large  nerve  bundles 
which   run   towards   the   surface. 

Soft  palate.    V.S.     Dog.     (p.  3,  s.  22  &  24,  e.  P.,  m.  B.)     (L) 

and     (H)      Recognise    the    mucosal     surfaces    covered    on    the 

nasal    side   by   stratified    ciliated,   and,    on   the   oral   aspect,    by 

stratified  squamous  epithelium.     Between  these  there  is  striped 

muscle    loosely    arranged     (Azygos    uvula?,     etc.)     and     mucous 

glands,    the  ducts   of   which   open   on   both   surfaces. 

The  preparations  of  the  remainder  (tubular  portion)  of  the  ali- 
mentary canal  which  follow  this  resemble  each  other  in  the  general 
plan  of  their  construction.  All  of  them  have  three  concentric  coats. 
(1)  Mucous,  (2)  Sub-mucous,  (3)  Muscular,  to  which  externally  a  fourth 
or  Serous  coat  of  varying  extent  is  added  in   the  peritoneal  cavity. 


70  ALIMENTARY     CANAL. 

The  mucous  coat  is  marked  off  from  the  sub-mucosa  by  a  layer  of 
non-striped  muscle  {muscularis  mucosce)  which  forms  a  continuous  covering 
from  the  stomach  downwards,  but  which  is  broken  up  into  strands  in 
the  oesophagus. 

The  muscular  coat  in  the  intestine  consists  of  an  outer  layer  with 
longitudinally-disposed  non-striped  fibres  and  an  inner  one  in  which  the 
distribution  is  circular.  Between  these  layers  is  found  the  richly  - 
ganglionated  nervous  plexus  of  Auerbach.  In  the  stomach  the  arrange- 
ment is  less  regular.  In  the  upper  part  of  the  oesophagus  striped  muscle 
is  substituted  (constrictors  of  the  pharynx). 

The  sub-mucosa  of  loose  areolar  tissue  allows  free  movement  to  the 
mucosa  when  the  latter  is  thrown  into  folds  by  the  contraction  of  the 
gut.  Through  it  blood  vessels,  nerves  and  lymphatics  (lacteals)  run  to 
their  points  of  distribution.  In  it  are  found  the  ganglia  of  the  plexus 
of  Meissner. 

Most  of  the  following  sections  will  in  all  probability  require 
to  be  flattened  on  warm  cedar  oil. 

(Esophagus.  T.S.  Dog.  (p.  8,  s.  17,  and  iodine  green,  c.  P., 
m.  B.)  The  mucosa  by  its  large  folds  practically  obliterates 
the  lumen  of  the  tube.  Its  free  surface  exhibits  stratified 
squamous  epithelium  supported  by  fibrous  tissue,  which  is 
compact  where  it  meets  the  epithelium.  In  this  tissue  the 
bands  of  muscularis  mucosae  seen  in  section  form  a  definite 
outline.  In  the  sub-mucosa  clusters  of  mucous  glands  occur 
at  frequent  intervals  and  their  wide  ducts  taper  to  a  small 
aperture  where  they  open  through  the  epithelial  covering.  The 
muscular  coat  is  non-striped  internally  and  of  the  striped 
variety  externally,  the  section  having  passed  through  the 
region  where  the  transition  occurs.  Examine  (//)  the  acini 
lined   with  secreting  epithelium  (green)  and   the   niuco-mucosse. 

aq  Stomach.  6 'ardio-cesoph a ujeal  junction.  V.L.S.  Cat.  (p.  3,  inj., 
s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  the  point  of  transition  of  the 
epithelium  of  the  oesophagus  into  the  mucosa  of  the  stomach. 


CARDIAC     ANI>     PYLORIC     STOMACH.  71 

(//)  The  former  layer  tapers  off  suddenly  to  meet  the  single 
row  of  columnar  epithelium  on  the  gastric  surface.  Recognise 
the  gastric  follicles,  tubular  glands  set  side  by  side,  the  length 
of  which  determines  the  thickness  of  the  membrane.  Observe 
the  muscularis  mucosa*  and  that  the  muscular  coat  is  not 
markedly    thickened    (Cardiac    sphincter  1 ). 

Cardiac  end  of  stomach.  V.S.  Kitten,  (p.  tfc  c.  the  same 
as  the  preceding.)  The  gastric  glands  not  being  fully  grown 
are  less  closely  packed  and  therefore  better  seen  individually 
than  in  the  adult  structure.  (L)  The  mucosa  is  the  thickest 
of  the  coats.  In  it  find  the  follicles  divisible  into  the  gland 
proper,  and  a  short  wide  duct  which  presents  a  wide  opening 
on  the  surface.  The  duct  is  lined  by  a  single  layer  of 
columnar  cells,  the  same  as  that  on  the  inner  surface  of  the 
organ.  The  gland  tubule  presents  two  kinds  of  epithelial 
cells,  the  inner  of  which  are  small  and  clear  and  almost  fill 
the  cavity  and  are  known  as  the  chief  cells.  Outside  are  the 
parietal  cells,  oval  nucleated  masses  of  protoplasm  which  cause 
lateral  projections  in  the  outlines  of  the  tubules.  In  the 
lower  part  of  the  mucosa  find  cross  sections  of  the  glands 
and  note  that  the  parietal  cells  also  project  inwards  between 
the  chief  cells  and  so  establish  a  connection  with  the  secretory 
passage.  The  interstitial  material  between  the  glands  consists 
of  fine  adenoid  tissue  in  which  blood-vessels  and  strands  from 
the    subjacent    muscularis    mucosae    should    be    found. 

Pyloric  mucosa.  V.S.  Kitten,  (p.  3,  inj.,  &c,  as  before.)  The 
secreting  portions  of  the  glands  are  lined  by  cells  similar  to  the 
chief  cells  ;  there  are  no  parietal  cells.  In  the  sub-mucosa  lymph 
nodules  are  frequently  met  with.  The  main  mass  of  these  lies 
in  this  layer;  a  portion,  however,  projects  into  the  mucosa  in 
a  somewhat  diffuse  manner. 


72  ALIMENTARY    CANAL. 

Pyloro  duodenal  junction.  V.L.S.  Cat.  (p.  3,  inj.,  &c.,  the  same 
as  the  last.)  (L)  Follow  the  surface  of  the  mucosa  from  the 
pyloric  side,  and  observe  that  the  transition  to  duodenum  is 
marked  by  the  gradual  appearance  of  projections  (villi)  in  the 
spaces  which  correspond  to  the  intervals  between  the  ducts  of 
the  gastric  glands.  The  mucosa  is  apparently  increased  in  thick- 
ness ;  this  is,  however,  due  to  the  compact  mass  of  glandular 
tissue  (Brunner's  glands)  in  the  sub-mucosa,  which  may  at  first 
be  taken  as  part  of  the  mucosa  until  the  position  of  the  muse, 
mucosae  is  recognised.  The  villi  are  column-like  projections  from 
the  mucosa  proper.  In  the  latter,  recognise  the  glands  of 
Lieberkiihn,  which  open  on  the  surface  between  the  villi.  They 
are  simple  tubular  glands.  The  muscular  coat  of  the  stomach 
undergoes  a  sudden  and  great  increase  to  form  the  pyloric  ring, 
which  diminishes  as  rapidly  on  the  duodenal  side  and  there 
presents  two  regularly  disposed  layers.  In  the  duodenum  lymph 
nodules  are  met  with  in  the  sub-mucosa.  Their  dome-like  pro- 
jections penetrate  to  the  free  surface  of  the  mucosa.  (See 
Peyer's  patch  later.) 

(H)  Examine: — -The  mucosal  transition;  the  villi;  the 
openings  of  the  Lieberkiihn's  glands  on  the  surface,  and  the 
epithelium  which  lines  them ;  also  the  ducts  and  secreting  acini 
of  Brunner's  glands,  the  epithelium  of  which  resembles  that  in 
mucous  glands  ;  the  muscular  coat  and  Auerbach's  plexus. 

Injected  stomach.  Y.S.  Fundus  of  cat's  stomach.  (Carmine 
gelatin  mass,  p.  2  {d),  s.  22,  c.  P.  or  G.,  m.  F.  or  B.)  Follow  the 
large  blood  vessels  from  the  sub-mucosa  to  their  branchings 
in  the  mucosa.  The  arteries  penetrate  nearly  to  the  surface 
before  dividing,  and  then  sub-divide  into  a  capillary  network 
which  forms  a  return  system  through  the  mucous  membrane 
around  the  glands  to  converge  into  a  system  of  veins  which 
debouch  into  the  sub-mucosa.  The  arteries  are  terminal,  e.g., 
have  no  free  anastomoses. 


SMALL     [NTESTINE.  73 

Small  Intestine.  For  the  structure  of  villi.  The  section 
contains  L.  and  T.S.  mounted  side  by  side.     (p.  3,  inj.,  s.  22  &  24, 

c.  P.,  m.  B.)  Recognise  (//)  the  single  layer  of  columnar  cells 
on  the  surface,  together  with  their  end  plates.  This  layer 
rests  on  a  well-defined  surface,  formed  by  the  adenoid  reticulum 
which  constitutes  the  supporting  framework  of  the  villus. 
Internal  to  this  find  blood  capillaries  (probably  quite  invisible 
being  collapsed)  and  next  a  zone  of  muscular  strands  from 
the  muse,  mucosae.  In  the  middle  the  central  lacteal  may  be 
observed  as  a  partially  open  cleft. 

>Small  intestine  for  fat  absorption.  Frog.  (The  animal  is 
fed  upon  a  piece  of  bacon  fat  or  some  lard  and  is  killed  four 
or  five  hours  afterwards.  The  intestine  is  cut  out  and  small 
pieces  are  quickly  placed  in  1  p'c-  osmic  acid  for  48  hours, 
p.  absolute  alcohol  with  eosin,  c.  P.,  m.  B.)  (77)  Find  the 
columnar  cells  loaded  with  small  fat  granules  near  their  free 
ends  which  become  larger  towards  the  nucleus.  Is  there  any 
fat  to  be  seen  in  the  substance  of  the  end  plate  1  Much  fat 
is  recognisable  in  the  subjacent  tissue  of  the  villus. 

Small  intestine  of  rabbit,  for   Auerbach's  plexus. 

A  loop  of  small  intestine  is  washed  out  with  normal  saline  and  is 
then  tied  at  one  end  and  filled  with  Ranvier's  boiled  gold  solution 
(28).  The  fluid  is  kept  in  by  a  second  ligature  and  the  whole  is 
immersed  in  gold  solution  for  4o  minutes.  The  gut  is  then  cut  into 
short  lengths,  is  rinsed  in  distilled  water,  and  is  transferred  to  20p-c-  formic 
acid  for  24  hours,  or  longer,  until  the  gold  is  reduced,  which  will  be 
recognised  by  the  uniform  reddish  violet  colouration.  Place  in  glycerin 
with  a  little  formic  acid  until  required. 

A  piece  of  the  gut  is  laid  open  by  a  longitudinal  incision  and  laid 
peritoneal  surface  downwards  upon  a  slide.  The  mucosa  is  next  scraped 
off  with  the  flat  of  a  needle  so  as  to  leave  only  the  muscular  coat,  m. 
in  glycerin  containing  a   little   formic   acid. 

(Z)  The  plexus  will  be  recognised  as  a   large  somewhat  square 

meshed    network    lying    between    the    layers    of    the    muscular 


74  ALIMENTARY    CANAL. 

tissue.      (H)  Recognise    the   nerve   cells   at    the   nodes.      They 
do   not   show  very  distinctly  as    a   rule. 

Peyer's  patch.  V.S.  Cat.  (p.  3,  inj.,  s.  22  &  24,  c.  P.,  m.  B.)  In 
the  sub-mucosa  find  the  clusters  of  lymph  nodules ;  these  project 
to  the  free  surface  of  the  mucosa,  where  they  are  covered  by  a 
single  layer  of  flattened  cells  which  (H)  exhibit  local  thinnings, 
so  that  the  line  of  separation  between  the  cell-loaded  adenoid 
tissue  and  the  cavity  of  the  gut  is  reduced  to  a  thin  film  with 
possible  apertures.  Between  the  rounded  heads  of  neighbouring 
nodules  the  villi  form  narrow  fringes. 

Injected  Peyer's  patch.  (Blue  gelatin  mass.  p.  2  {d),  s.  24, 
c.  P.,  m.  B. ;  or  c.  G.,  s.  24  on  the  slide,  m.  B.)  (L)  The  injected 
capillaries  form  a  network  in  the  villi  close  under  the  epithelium, 
and  are  connected  with  vessels  in  the  sub-mucosa.  "  Capillary 
loops,  few  in  number,  project  from  the  surface  into  the  lymph 
nodules,  which,  though  rich  in  cells,  are  not  very  vascular.  In 
this  respect  they  resemble  lymphatic  glands.     (See  later.) 

Large  Intestine.  V.S.  Cat  or  Dog.  (p.  3,  inj.,  s.  22  &  24, 
c.  P.,  m.  B.)  (L)  There  are  no  villi,  and  the  only  glands  are  of 
the  Lieberkiihn  variety.  Lymph  nodules  occur  occasionally. 
The  muscular  wall  is  thinner  than  that  of  the  small  intestine. 
(II)  Goblet  cells  are  numerous  in  the  glands  amongst  the 
other  cells. 

Injected  large  intestine.  (Blue  gelatin  mass.  p.  2  (rf),  s.  24,  c.  P., 
m.  B.)  The  blood  vessels  form  a  rich  capillary  network  around 
the  glands. 

Vermiform    Appendix.    Man.    T.S.    (p.  3,  s.  22  &  24,  c.  P., 

m.  B.)  (I)  The  mucosa  is  scantily  supplied  with  Lieberkuhn's 
glands ;  there  is  much  interstitial  tissue  in  which  lymph  nodules 
are  somewhat  diffusely  projected  from  the  sub-mucosa,     (II)  The 


VERMIFORM     APPENDIX.  75 

latter  is  of  comparatively  dense  fibrous  tissue  in  which  lymph 
-paces  occur,  the  endothelial  lining  of  these  is  often  well  seen. 
The  muscular  coat  is  unusually  thick  as  compared  with  that  of 
other  parts  of  the  intestine. 


CHAPTER  XL 
GLANDS  OF  THE  ALIMENTARY  CANAL. 

Most  of  the  sections  of  glands  will  require  to  be  fixed  to  the 
slide  by  means  of  shellac  fixative  very  thinly  applied. 

Parotid  Gland  (Serous)  Cat.  (p.  3,  inj.,  s.  21,  c.  P.,  m.  B.) 
(L)  Small  lobules  separated  by  loose  inter-lobular  connective 
tissue.  The  acini  are  of  rather  uniform  appearance,  and  can 
be  recognised  under  this  power.  The  ducts  and  their  branchings 
are  obvious  inside  and  between  the  lobules,  and  are  accompanied 
by  blood-vessels.  (H)  The  acini  are  filled  with  redundant 
secreting  epithelial  cells  of  the  serous  variety.  Their  nuclei 
are  placed  near  the  periphery  of  the  cell,  and  the  cytoplasm 
is  finely  reticulated.  Acquaint  your  eye  with  the  general 
appearance  of  these  cells.  Find  the  commencement  of  ducts 
in  the  acini,  at  first  lined  by  flattened  cells  to  change  to  cubical 
and   even   columnar   forms   in   the  larger  canals. 

Sub-maxillary  Gland.  Dog.  (Mucous.)  (p.  8,  inj.,  s.  22  &  24, 
c  P.,m.B.)  (L)  The  lobules  are  more  compact  than  in  the  last, 
the  acini  larger,  and  the  ducts  less  numerous.  (H)  The 
secreting  epithelium  is  larger,  the  cytoplasm  less  reticular  and 
very  transparent  in  appearance,  and  the  nucleus  is  pressed 
against  the  attached  surface  of  the  cell.  Recognise  the  crescentic 
cells  or  demilunes  (Gianuzzi)  placed  outside  the  mucous  cells,  and 
which  occasionally  jut  inwards  between  them.  Their  cytoplasm 
is  delicately  reticulated,  resembling  the  substance  of  the  serous 
cell,  and  the  nucleus  is  rounded.  The  ducts  are  lined  by  cubical 
epithelium,  the  larger  ones  being  accompanied  by  blood-vessels 
and   ganglionated   nerves. 


SALIVARY    GLANDS. PANCREAS.  77 

Sub-maxillary  Gland.  Man.  (Muco-serous.)  (p.3,s.21,c.P.,m.B.) 
(Z)  The  mucous  acini  appear  like  perforations  amongst  the 
more  solid  serous  ones.  In  many  lobules  the  larger  ducts 
(columnar  epith.)  are  surrounded  by  dense  fibrous  tissue,  in 
which  blood-vessels  and  nerves  are  also  placed.  (H)  Observe 
the  character  of  the  epithelium  in  the  two  varieties  of  acini. 
In  the  serous,  the  nuclei  are  round  and  are  placed  near  the 
middle  of  the  cell. 

Salivary  gland  injected.  Kitten.  (Blue  gelatin  mass, 
p.  2  {d),  s.  17,  c.  P.,  m.  B.)  (L)  There  is  a  rich  distribution  of 
capillaries  in  the  lobules  and  in  the  walls  of  the  large  ducts. 
Note  also  masses  of  fat  injected.  (H)  The  capillary  networks 
lie  close  upon  the  surface  of  the  acini  and  around  the  fat  cells. 
Paccinian  corpuscles  occur  in  the  gland  substance  in  this 
animal.  Observe  that  capillaries  penetrate  to  the  interior  of 
the    corpuscle   and    follow  the   nerve   to   its    termination. 

Pancreas.      Rat.      (p.  4,  s.  22  &  24,  c.  P.,  m.  B.)     (L)    The   21 

lobules  are  frequently  fusiform  from  mutual  compression  and 
the  connective  tissue  between  is  scanty.  Ducts  have  thin 
walls  and  are  not  prominent.  (H)  Acini  are  rather  narrow, 
the  epithelium  is  dense  looking,  and  the  cytoplasm  is  divided 
into  an  outer  hematoxylin  coloured  layer  and  an  inner  eosin 
stained  granular  portion.  These  granules  are  characteristic 
(Zymogen).  Find  the  interalveolar  cell  islets,  irregularly  shaped 
clusters  of  polygonal  cells  devoid  of  eosin  stained  granules. 
They  occur  in  the  substance,  most  commonly,  or  in  the  neigh- 
bourhood of  the  ducts.  Their  general  appearance  in  stained 
sections  is  paler  than  that  of  the  surrounding  tissue,  they  can 
therefore  be  recognised  under  a  low  power.  The  interlobular 
blood-vessels    are   accompanied    by  ganglionated    nerves. 


78  GLANDS  OF  THE  ALIMENTARY  CANAL. 

Liver-  Liver  cells.  A  scraping  from  the  cut  surface  of  the 
fresh  liver  of  a  mammal,  diffused  in  salt  solution.  (H)  Sketch 
their    shape   and    contents. 

Liver  for  Glisson's  capsule.  Pig.  (p.  3,  inj.,  c.  G.,  s.  19,  m.  F.) 
(L)  Externally  the  capsule,  internally  the  polygonal  lobules 
separated  from  each  other  by  interlobular  extensions  of  Glisson's 
capsule.  Find  a  lobule  with  a  central  vessel,  note  the  radiating 
arrangement  of  the  lobular  substance.  The  portal  vein  is 
distributed  in  the  capsular  tissue  and  is  accompanied  by  one 
or  more  branches  of  the  hepatic  duct  and  artery  which  together 
constitute    a  portal    tract. 

The  hepatic  vein  springs  in  the  centre  of  the  lobule.  Find 
examples  of  it  cut  across  and  lengthways.  The  ^  latter  can 
be  traced  into  the  capsular  tissue.  Two  or  more  of  them  may 
be  found  converging  from  contiguous  lobules  to  a  larger  hepatic 
vein  (sub-lobular  vein).  (H)  The  free  surface  is  covered  by 
the  capsule  of  dense  white  fibrous  tissue  with  distinct  corpuscles 
and  numerous  small  lymph  spaces.  Study  the  hepatic  cell 
substance.  It  forms  a  columnar  network,  usually  two  cells 
thick,  interlaced  with  the  system  of  capillaries.  The  liver  cells 
are  nucleated  and  have  a  reticulated  cytoplasm  often  vacuolated. 

Liver.  Rabbit,  for  portal  tract*  and  cell  columns,  (p.  6, 
s.  22  &  24,  c.  P.,  m.  B.)  (L)  In  a  portal  tract  recognise  the 
large  thin-walled  portal  vein.  The  small,  thick-walled  hepatic 
duct,  lined  with  cubical,  or  even  columnar,  epithelium,  and  the 
small  hepatic  artery.  There  is  no  corresponding  vein,  the 
blood  return  taking  place  through  the  hepatic  vein.  (II)  Con- 
firm the  above  and  observe  that  the  lobular  capillaries  open 
into  the  hepatic  vein.  The  cell  columns  are  everywhere  in 
contact  with  capillaries,  the  endothelial  covering  of  which  is 
recognised  by  their  nuclei  seen  in  profile. 


BILE    DUCTS. — GLYCOGEN.  79 

Liver.  Man,  stained  Golgi  for  bile  ducts,  (p.  11,  s.  27,  c.  G., 
m.  B.,  uncovered,  or  see  11.)  (//)  Find  the  black,  close,  and 
angular  meshed  system  of  bile  channels  amongst  the  cells. 
They  open  into  the  ducts  in  the  capsular  connective  tissue. 

Liver.  Kitten,  for  larger  ducts  and  gall  bladder,  (p.  3,  inj., 
s.  22  &  24,  o.  P.,  m.  B.)  (L)  Find  a  large  duct  in  a  portal 
tract.  The  lining  epithelium  is  cubical  and  thrown  into  folds. 
Externally  to  this  observe  the  small  sacculations  lined  with 
the  same  kind  of  epithelium,  so-called  mucous  glands,  the 
epithelium  of  which,  however,  does  not  resemble  that  of 
mucous  glands  elsewhere.  Examine  the  wall  of  a  portal  vein 
and  note  that  there  is  hardly  any  muscular  tissue  in  it.  The 
gall  bladder  is  lined  by  cylindrical  cells.  Note  the  glandular 
sacculations.  Outside  this  find  the  pronounced  muscular  coat 
containing  ganglionated  nerves. 

Liver.  Rabbit,  for  glycogen,  (p.  Absolute  alcohol  injected, 
c  P.)  After  the  removal  of  the  paraffin  on  the  slide,  stain  with 
a  strong  alcoholic  solution  of  iodine  in  iodide  of  potassium,  m.  B. 
The  brown-red  stain  brings  into  view  the  nodular  masses  of 
the   glycogen  in  the  cytoplasm. 


CHAPTER    XII. 
KIDNEY. 

Kidney,  of  a  small  mammal  for  the  general  arrangement  of 
the  tubules.  T.S.  Rat.  (p.  3,  inj.,  s.  22  &  25,  c.  P.,  m.  B.)  (L) 
Externally  the  thin  and  easily  detached  capsule  rests  upon  the 
cortex,  the  latter  is  readily  distinguished  from  the  medullary  or 
central  portion  of  the  organ  by  its  pinker  stain.  The  medulla 
projects  inwards  as  a  conical  papilla.  The  clear  space  around 
the  latter  is  the  sinus  of  the  kidney,  lined  by  the  pelvic  expansion 
of  the  ureter,  a  section  of  the  latter  will  be  found  in  the  mass  of 
fat  immediately  outside.  The  cortex  nearly  meets  around  the 
medullary  part  at  this  level  of  the  organ.  A  little  way  above  or 
below  the  cortex  would  be  continuous  all  round.  Just  outside 
the  pelvic  membrane,  find  the  Renal  arteries  and  the  larger  thin- 
walled  veins. 

The  medullary  rays  (pyramids  of  Ferrein)  are  clusters  of  renal 
tubules  which  run  outwards  from  the  medulla  into  the  cortex, 
where  they  gradually  disappear.  The  intervals  between  them 
are  filled  by  the  convoluted  tubes  which  exhibit  the  pink  stain, 
and  amongst  which  note  the  bluer  coloured  glomendi. 

Kidney,  for  T.S.  papilla.  Rabbit,  (p.  4.,  s.  17,  c.  P.,  m.  B.) 
(L)  Find  the  papilla  and  recognise  near  its  apex  large  collect- 
ing tubes  (ducts  of  Bellini),  and  if  the  section  favours  their 
openings  on  the  surface.  Tf  cut  lengthways  their  dichotomous 
divisions  will  be  found.  Between  them  (//)  there  is  much 
interstitial  tissue,  in  which  the  capillaries  are  recognisable  by 
their  thin  endothelial  coverings.  Towards  the  cortex  the 
loops   of  Henle's  tubes  are   to  be  found. 


BOUNDARY    ZONE. BLOOD    VESSELS.  81 

Kidney.  Dog,  for  boundary  zone.  (p.  12,  s.  22  &  24,  c.  P., 
m.  B.)  (L)  Between  the  cortex  and  medulla  find  the 
boundary  zone,  rendered  distinct  from  either  of  these  by  the 
closely  packed  parallel  tubes,  chiefly  ascending  and  descending 
limbs  of  Henle's  loop.  Observe  the  large  blood-vessels  on  a  level 
with  this  zone.  (H)  Find  loops  at  different  levels  in  this  stratum, 
and  note  the  character  of  the  epithelium  with  which  they  are 
lined.  Find  a  glomerulus,  cut  through  its  attachment  to 
Bowman's  capsule;  observe  that  the  former  is  compounded  of 
clusters  of  capillaries,  and  also  that  its  surface  is  covered  by  a 
layer  of  epithelium,  the  reflected  part  of  the  capsule,  the  nuclei 
of  which  are  superficial  to  those  of  the  capillaries.  Search  for 
the  connection  of  the  capsule  with  a  convoluted  tube,  and  inspect 
its  epithelium.  Next  note  the  shape  of  the  cells  in  the  collecting 
tubes  of  the  medullary  rays,  and  compare  them  with  those  of 
Henle's  loops  in  the  boundary  zone. 

Injected  Kidney  of  a  mammal.  Cat.  (Blue  or  red  gelatin 
mass,  s.  17  or  22,  c.  G.  or  P.,  m.  B.  or  F.)  (L)  Find  the  arching 
arteries  and  veins  in  the  intermediate  zone  and  trace  their  two- 
fold distribution.  (1)  Outwards  to  the  cortex,  the  interlobidar 
arteries  give  off  efferent  branches  to  the  glomeruli,  the  rich 
cluster  of  capillaries  wThich  form  the  latter  and  the  efferent 
ve-sel  which  leaves  them  to  divide  into  a  second  set  of  capil- 
laries, the  intertubular  plexus  of  capillaries.  The  venous  return 
from  these  occurs  through  the  interlobular  veins,  which  com- 
mence on  the  surface  of  the  cortex  as  the  stellate  veins.  From 
the  lower  glomeruli  the  efferent  vessel  forms  a  leash  of 
capillaries,  pseudo-arterice,  rectce,  which  pass  into  the  medulla. 
C2)  Inwards  branches  break  up  at  once  into  parallel  clusters 
of  vessels,  the  arterice  rectce,  which  return  their  blood  through 
a   similar   set   of  small   vessels   into   the   arching  veins. 


82  KIDNEY. 

Isolated  renal  tubules  of  small  rabbit,  (p.  slices  in  30 cc  HC1 
for  2  or  3  hours.  Dissociate  the  renal  substance  into  wedges, 
and  separate  by  agitation  in  water)  (Stohr).  (After  thorough 
washing  stain  48  hours  in  19  and  dissociate  in  glycerin  jelly; 
m.  in  the  same).  Find  glomeruli  in  their  Bowman's  capsules 
connected  with  convoluted  tubes,  fragments  of  Henle's  loops  and 
of  Collecting  tubes.  The  character  of  the  epithelium  is  not  very 
much  altered. 

JEmbyonic  Kidney,  from  a  mammalian  Foetus,  preferably 
human.  T.S.  (p.  15,  s.  17  or  22,  c.  P.,  m.  B.)  (L)  The  tubules  are 
simple  and  visible  throughout  their  whole  extent;  they  run 
nearly  straight  from  the  papilla,  and  have  a  crook-shaped  bend 
close  to  the  cortical  surface,  where  they  present  a  cup-shaped 
expansion.  There  is  much  intestitial  connective  tissue.  (H) 
Examine  the  peripheral  expansion  of  the  tube  (early  stage  of 
Bowman's  capsule),  recognise  its  crescentic  form,  the  concavity  of 
which  is  made  up  of  tall  cells,  and  partially  embraces  the  future 
glomerulus.  At  this  stage  the  glomerulus  appears  as  a  mass  of 
cells  connected  with  the  blood-vessel.  The  details  vary  in  their 
form  with  the  state  of  the  development  of  the  individual 
glomerulus.  In  the  young  compound  kidney  (human),  the 
individual  malpighian  pyramids  or  simple  kidneys  are  in  contact 
along  the  lateral  portions  of  their  cortices  (columns  of  Bertin). 

Ureter.  Kitten  or  Dog.  T.S.  (p.  3,  inj.,  s.  22  &  24,  c.  P., 
ni.  B.)  (L)  The  mucous  coat  is  covered  with  stratified  epithelium 
of  the  transitional  variety ;  note  the  flattened  superficial  cells. 
Examine  the  loose  connective  coat  outside,  and  observe  that  it  is 
vascular.  External  to  this  is  the  muscular  coat,  the  strongest 
part  of  which  is  transversely  disposed,  longitudinal  fibres 
occurring  on  both  its  inner  and  outer  aspect.  Externally  to  all 
is  a  loose  fibrous  covering. 


URINARY    BLADDER.  83 

Bladder  Kitten.  T.S.  (p.  3,  inj.,  s.  22  &  24,  c.  P.,  m.  B.) 
(Z)  The  mucous  coat  resembles  that  of  the  ureter,  but  is  thrown 
into  greater  folds.  The  muscular  coat  is  thicker,  and  the  con- 
stituent fibres  are  massed  into  two  or  three  layers.  (H)  Note 
capillaries  in  the  connective  tissue,  just  outside  the  transitional 
epithelium. 


CHAPTER   XIII. 

SKIN   AND   ITS   APPENDAGES. 

Skin.  Palmar  surface  of  human  finger,  (p.  11,  s.  22  &  24, 
c.  P.,  m.   B.) 

(L)  The  epidermis  has  already  been  described.  (See  stratified 
epithelium. ) 

The  dermis  or  true  skin  consists  of  fibrous  tissue  of  con- 
siderable density  near  the  surface,  but  looser  deeper  down, 
where  also  are  masses  of  sub-cutaneous  fat  (paniculus  adeposus). 

The  papillce  are  buried  in  the  epidermis  and  contain  vascular 
loops  or  nerve  endings  (touch  corpuscles)  sometimes  in  the 
same  papilla,  but  usually  in  separate  ones.  These  corpuscles 
are  masses  of  an  ovoid  shape  consisting  of  spirally  wound  fibrous 
tissue  in  which  a  nerve  may  be  seen  to  end.  The  sweat  glands 
are  simple  tubes,  the  secreting  portions  of  which  lie  deep  in  the 
dermis,  where  each  forms  a  convoluted  mass.  From  these  the 
duct  runs  to  the  surface,  passing  through  the  epidermis  in 
a  spiral  course.  Find  on  a  level  with  the  fat  the  paccinian 
bodies.  In  structure  they  are  concentrically  laminated  around 
the  nerve,  of  which  they  form  the  terminal  coverings.  Here 
find,  also,  small  arteries  and  nerve  bundles.  (//)  Examine 
the  structure  of  the  touch  corpuscles.  The  secreting  part 
of  the  sweat  glands  in  T.S.  shows  externally  a  hyaline  mem- 
brane, next  to  this  a  layer  of  longitudinally  disposed  non- 
striped  muscle,  and  innermost  of  all  secreting  epithelium. 
The  ducts  are  lined  by  low  cubial  cells  for  which  the 
squamous  variety  is    substituted    in    the   epidermis. 


SCALP.  —  NAIL.  85 

Scalp,  V.S.  Human,  (p.  11,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  The 
hair  follicles  which  are  set  aslant  consist  of  a  central  hair  (shaft 
and  bulb)  closely  invested  by  the  inner  and  outer  root  sheaths 
of  epithelium,  which  are  respectively  continuous  with  the  Horny 
layer  and  Rete  mucosum  of  the  surface.  Trace  the  connection. 
Covering  these  is  the  thin  Dermic  coat,  a  surface  condensation 
of  fibrous  tissue.  The  sebaceous  glands  form  compound  saccu- 
lated masses  outside  the  follicles,  into  which  their  ducts  open 
not  far  from  the  surface.  Further  down  find  the  Erector  Pili, 
a  band  of  non-striped  muscle  which  stretches  across  the  obtuse 
angle  formed  by  the  follicle  with  the  surface  of  the  skin. 
Observe  its  method  of  attachment  at  both  ends.  (//)  Examine 
the  constitution  of  the  root  sheaths,  and  trace  them  to  the  bulb 
and  over  the  vascular  papilla  situated  within  it.  Make  out 
the  formation  of  the  hair  at  its  bulb  from  the  epithelial  cells  of 
the  sheaths.  Examine  the  secreting  cells  of  the  glands ;  the 
lumen  is  filled  with  cells,  which  break  up  into  granules  of 
sebaceous  material. 

Scalp,  human,  horizontal  S.  (p.  the  same  as  the  preceding.) 
Examine  the  root  sheaths  of  the  hair  follicles  especially,  and 
the   other   features   previously  mentioned. 

Nail.  Human  foetal  finger.  V.L.S.  (p.  15,  s.  22  &  24, 
c.  P.,  m.  B.)  (L)  On  the  dorsal  aspect  of  the  finger,  and  near 
its  tip,  find  the  obliquely  placed  invagination  of  epidermis  in 
which  the  root  of  the  nail  is  buried.  At  this  early  stage 
the  nail  appears  little  more  than  a  thickening  of  the  horny 
layer.  A  T.S.  is  required  to  show  the  mucosal  ridges  which 
only  become  marked  later.  Note,  besides,  in  other  parts  of  the 
skin,  the  developing  siveat  glands,  which  may  only  be  present 
as  columnar  invaginations  of  the  rete  mucosum.  Examine 
also  the  devoloping  phalanges  with  their  cartilaginous  ends 
and  the  marrow  in   their   interior. 


CHAPTER   XIV. 
BLOOD    GLANDS. 

Lymph  gland.  T.S.  (p.  8,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  The 
organ  is  divided  into  an  external  denser  cortex  and  a  more 
open  inner  texture,  the  medulla.  The  latter,  especially  in 
small  glands,  is  placed  close  to  one  side  near  the  hykun,  the 
place  of  entrance  of  the  blood-vessels.  A  capsule  covers  the 
surface  from  which  prolongations  the  trabecular  run  inwards 
nearly  straight  in  the  cortex,  to  become  network-like  in  the 
medulla.  In  small  glands  this  tissue  is  scanty.  A  narrow, 
clear  space,  the  lymph  sinus,  separates  them  everywhere  from 
the  follicular  portion,  the  substance  proper  of  the  organ. 
In  the  cortex  there  are  frequently  rounded  areas  rendered 
evident  by  the  stain,  and  which  bear  a  close  resemblance  to 
the  splenic  bodies.  (H)  The  capsule  is  of  fibrous  tissue  and 
contains  varying  quantities  of  non-striped  muscle.  The  sinus 
is  traversed  by  branching  lymphoid  tissue  with  scattered  cells 
in  its  meshes  (lymph  cells).  The  follicular  tissue  is  sharply 
marked  off  from  the  sinus,  and  is  packed  with  lymph  cells, 
the  nuclei  of  which  show  frequent  evidence  of  mytosis. 

Injected  lymph  gland.     (Blue  gelatin  mass,  p.  2  {d),  s.  24,  c.  P., 
m.   B.)      The  blood-vessels  enter   at   the   hylum  and   ramify  in 
he  trabecule  to  be  distributed  in  capillary  loops  in  the  follicular 
system.     The  gland  is  not  very  vascular. 

Tonsil,  Dog.  V.S.  (p.  8,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  Find  the 
indented  oral  surface  covered  with  stratified  squamous  epithelium, 
and  beneath  this  the  lymph  nodules  embedded  in  areolar  tissue. 


H^MOLYMPH    GLANDS. — SPLEEN.  87 

There  are  also  mucous  glands  whose  ducts  open  surepficially.  (H) 
Examine  the  epithelium  over  the  nodules,  and  note  that  it  thins 
down  frequently  to  a  single  cell  over  the  lymph  nodules,  the  cells 
of  which  may  easily  pass  through  such  places. 

Hsemolymph  glands,  from  the  prevertebral  fat  of  the  sheep.    24 
(p.  3,  s.  22  &  24,  c.  P.,  ra.  B.)    These  structures   closely  resemble 
small  lymph  glands,   the  notable  difference  being  that  the  wide 
sinuses,  which  are  particularly  evident  in  the  medulla,  are  filled 
with  red  blood-cells  instead  of  lymph  corpuscles. 

Spleen,  for  capsule  and  trabecule,  Sheep,  (p.  8,  s.  22  &  24, 
c  P.,  m.  B.)  (L)  The  muscular  capsule  is  thick,  and  sends 
trabecule  into  the  interior  of  the  organ.  The  pulp  lies  close  to, 
and  is  in  contact  with  them  everywhere.  The  larger  trabecule 
are  tunnelled  by  blood-vessels.  The  pulp  is  uniform  in  appear- 
ance, excepting  where  it  presents  circular  and  more  darkly  stained 
outlines  in  its  substance,  the  splenic  bodies.  (H)  Examine  the 
capsule  and  trabecule  for  non-striped  muscle.  Follow  the 
trabecule  into  the  pulp,  note  the  vessels  in  their  interior  and 
then  inspect  the  pulp.  Little  more  can  be  made  out  beyond  the 
fact  that  it  is  crowded  with  cells.  The  outlines  of  the  splenic 
bodies  present  the  appearance  of  faint  concentric  striations. 

Spleen.  Kitten,  (p.  3,  inj.,  s.  22  &  24,  c.  P.  very  thin  m.  B-) 
(H)  Examine  in  the  pulp  for  the  commencement  of  veins.  These 
will  be  found  with  careful  search,  as  small  often  branching  spaces 
with  a  continuous  endothelial  outline  where  the  wall  is  complete, 
and  elsewhere  an  indefinite  boundary,  where  the  cavity  of  the 
vessel  merges  into  the  spaces  of  the  adenoid  tissue.  Many  of 
the  cells  are  loaded  with  coarse  granules  stained  with  eosin, 
which  form  mulberry-shaped  masses.  These  are  red  blood-cells 
in   course  of  disintegration. 


88  BLOOD    GLANDS. 

S])leen,  injected.  Cat.  (Blue  gelatin  mass.  p.  2  (d),  s.  24,  c  P., 
m.  B.)  (Z)  Trace  the  blood-vessels  through  the  trabecular  into 
the  splenic  bodies  and  observe  that  the  arterioles  penetrate  to 
their  interior,  give  off  a  few  capillary  branches  which  run  to  the 
surface  of  the  bodies,  and  there  discharge  their  contents  into  the 
general  space  of  the  pulp.  The  splenic  bodies  thus  form  rounded 
islands  of  almost  non-vascular  pulp,  surrounding  the  terminations 
of  the  arteries.     (Peri-angial  lymphatic  nodules?) 

Thyroid.  Man  or  Sheep,  (p.  10,  s.  Saffranin  in  aqueous  sol., 
c.  P.,  m.  B.)  (L)  The  organ  is  lobulated  with  loose  connective 
tissue  between.  The  spherical  vesicles  which  make  up  the 
substance  proper  of  the  gland  are  lined  by  cubical  epithelium, 
the  nuclei  of  which  are  visible  with  this  power.  The  vesicles 
are  filled  with  a  structureless  substance,  Colloid.  (H)  'Examine 
the  epithelium  and  the  disposal  of  the  colloid  which  can  be 
frequently  traced  between  its  cells.    Detached  cells  in  the  colloid. 

Thymus.  Child,  (p.  12,  s.  22  &  24,  c  P.,  m.  B.)  (L)  The  lobules 
are  separated  by  scanty  areolar  tissue.  The  substance  proper 
looks  uniformly  granular  with  the  exception  of  small  round  islands 
stained  pink,  HassalVs  corpuscles.  (H.)  The  substance  proper 
consists  of  delicate  adenoid  tissue  crowned  with  cells.  The  small 
Hassall's  corpuscles  are  nests  of  concentrically  packed  flattened 
cells.  The  larger  ones  exhibit  the  same  arrangement  at  their 
periphery,  whilst  the  interior  is  full  of  larger  rounded  cells,  the 
outlines  of  which  are  not  always  very  distinct.  These  bodies  are 
regarded  as  hypoblastic  relics. 

Thyroid,  parathyroid  and  thymus  of  Kitten,  (p.  3,  s.  22  &  24, 
c.  P.,  m.  B.)  (L)  The  partially  developed  thyroid  shows  besides 
the  vesicles  masses  of  interstitial  cells  in  which  the  gradations  of 
vesicular  formation  are  recognisable.  Small  quantities  of  colloid 
appears  amongst  the  cells,  which  are  gradually  thrust  apart  and 


PITUITARY    AND    SUPRARENAL    BODIES.  89 

expanded  int<>  vesicles  by  its  accumulation.  The  thymus  has 
already  been  described.  The  parathyroid,  distinct  from  either  of 
the  two  preceding  structures,  is  situated  between  them  at  this 
point,  and  consists  of  closely  packed  columns  of  epithelium,  which 
look  very  much  like  -land  tubules.  This  body  is  of  considerable 
vascularity,  there  being  many  capillaries  around  the  tubules. 

Pituitary  body.    Rabbit,    (p.  3,  s.  22  &  24,  c.  P.,  m.  B.)    The   25 

organ  is  surrounded  by  a  capsule  of  considerable  thickness,  and 
consists  of  two  portions.  The  anterior  is  developed  from  the 
dorsal  aspect  of  the  pharynx,  and  the  posterior  from  the  ventral 
aspect  of  the  brain.  The  anterior  portion  is  made  up  of  almost 
solid  columns  of  cells,  in  which  there  is  occasionally  the  appear- 
ance of  a  lumen.  These  columns  are  separated  by  loose  connective 
tissue.  Near  the  hinder  end  of  this  portion  duct-like  tubules, 
lined  by  cubical  cells  occur,  next  to  which  large  venous  spaces 
are  found.  The  latter  are  accompanied  by  small  lymph 
nodules.  The  posterior  portion  consists  of  fibrous  material, 
mostly  non-medullated  nerve  fibres,  amongst  which  there  are 
scattered  nerve  cells. 

Suprarenal  body.  T.S.  Man.  (p.  11,  s.  22  &  24,  c.  P., 
q.m.  B.)  (X)  A  thin  fibrous  capsule  surrounds  the  organ,  the 
interior  of  which  is  divided  into  cortex  and  medulla.  The 
latter  occupies  but  little  of  the  whole  organ,  and  is  of  a  more 
open  texture.  (7/)  Recognise  the  vertical  columns  of  cells  of 
which  the  cortex  is  composed,  they  present  a  somewhat  glan- 
dular appearance.  In  the  outer  part  of  the  cortex,  which  is 
known  as  the  glomerular  zone,  the  columns  are  wider  than  in 
the  inner  or  reticular  zone,  which  forms  the  mass  of  the  organ, 
and  the  columns  of  cells  do  not  intercommunicate  to  the 
same  extent. 


90  BLOOD    GLANDS. 

The  medullary  portion  presents  a  network  of  intercom- 
municating cell  columns,  the  interstices  of  which  are  occupied 
by  venous   channels. 

Suprarenal  body.  Kitten.  (Injected  carmine  gelatin,  s.  22, 
o.  P.,  m.  B.)  (Z)  and  (R)  Note  the  rich  capillary  distribution 
around  the  cell  columns  and  the  convergence  of  these  into 
the   larger   veins   of   the   medulla. 


CHAPTER   XV. 
NERVOUS    SYSTEM. 

Superior  cervical  gang-lion.     Man.     (p.  3,  s.  22  &  24,  c.  P., 

m.  B.)  (L.)  and  (H.)  Superficially  a  thin  laminated  perineurial 
sheath  internally  nerve  cells  of  various  sizes,  and  many  contain 
crescentic  patches  of  pigment  granules.  Each  cell  has  a 
nucleated  capsule.  There  is  much  tissue  between  the  cells 
consisting  of  non-medullated  nerve  fibres  and  filaments  and 
capillaries.  The  entering  nerves  are  of  the  non-medullated 
variety  for  the  most  part. 

Posterior  root  ganglion.      Man.      (p.  3  or  11,  s.  22  &  24, 

c.  P.,  m.  B.)  (L.)  and  (H.)  The  structure  is  fusiform,  and  there 
is  an  entering  and  an  emerging  mass  of  medullated  nerve  fibres. 
The  perineurial  sheath  is  well  marked.  The  cells  are  somewhat 
larger  than  in  the  sympathetic  and  of  a  more  uniform  size  ; 
their  capsules  are  distinct,  but  there  is  less  intervening  tissue. 

Vagus  Nerve,  Man  or  Dog,  T.S.  of.  In  the  neck.  (p.  Osmic 
acid,  c.  P.,  m.  B.)  The  perineurial  coat  is  strong  around  the  sub- 
divisions of  the  nerve.  Sketch  the  structure,  and  pay  special 
attention  to  the  size  of  the  nerves  in  the  fibres  in  different 
localities.     Compare  with  the  sciatic  nerve  examined  earlier  on. 

Spinal  Cord. 
Cord  of  rabbit.     T.S.  upper  thoracic  region,      (p.  3.,  s.  aqueous 
methylene  blue  in  bulk,  e.  P.,  m.  B.) 

Cord,  Man,  Thoracic  region  (p.  11,  s.  19,  c.  G..  m.  F. )  Examine 
both  of  these  for  the  following  points  of  structure.  (L)  The 
cord     is    closely     invested     by     the    thin     fibrous    pia     mater. 


92  NERVOUS    SYSTEM. 

Recognise  the  subdivision  of  the  cord  into  lateral  halves  by 
the  ventral  fissure  (open)  and  the  dorsal  fissure  (filled  with 
pia  mater),  which  dip  in  as  far  as  the  commissure  that  unites 
the  lateral  halves.  Within  the  white  matter  of  the  cord  lies 
the  grey  substance,  the  outline  of  which  forms  an  irregular 
letter  H,  being  united  across  the  middle  line  by  the  grey 
commissure.  Each  half  of  the  grey  matter  presents  a  narrow 
ventral  horn  which  terminates  in  a  square  end  some  distance 
from  the  free  surface. 

The  dorsal  horn,  slight  and  fusiform  in  shape,  attains  to  the 
surface,  where  it  is  met  by  the  dorsal  root  of  the  spinal  nerve. 
Nearly  midway  on  the  outer  side  of  the  grey  substance  the 
lateral  horn  forms  a  pointed  projection. 

The  white  substance  is  divided  into  three  main  anatomical 
subdivisions  : — (1)  The  ventral  column,  between  the  ventral  fissure 
and  the  outer  strands  of  the  ventral  nerve  root  fibres ;  (2)  The 
latercd  column,  extending  from  this  to  the  dorsal  root ;  and  (3) 
The  dorsal  column,  between  the  dorsal  root  and  the  corresponding 
fissure.  The  only  other  subdivision  of  the  white  substance  is 
occasionally  caused  by  a  thin  septum  of  pia,  which  separates 
the  dorsal  column  into  an  inner  G oil's  (Gracile)  column  and 
an  outer  Burdach's  (Cuneate)  column.  (H)  Examine  the  cells 
of  the  ventral  horn.  Search  for  their  axons  running  into  the 
strands  of  the  corresponding  nerve  root.  The  large  nerve  cells 
are  mostly  confined  to  this  cornu.  Inspect  the  lateral  cornu, 
in  which  small  nerve  cells  can  be  recognised  (Intermedio  lateral 
tract).  Dorsally  to  this  find  the  lateral  reticular  formation, 
a  network  of  fibres  passing  into  the  white  matter.  The  dorsal 
cornu  is  pointed  and  capped  by  the  gelatinous  substance  of 
Rolando.  The  dorsal  root  enters  its  apex  in  part  only,  most 
of  it  making  a  sweep  inwards  to  its  mesial  side.  On  the 
mesial   aspect  of   the  neck  of   the   dorsal    horn   near  the  com- 


SPINAL    CORD.  93 

missure  a  distinct  group  of  cells  (Clarke*  posterior  vesicular 
column)  will  be  seen,  if  the  section  be  low  enough  in  the 
thoracic  region.     The  cells  have  an  oval  outline. 

The  grey  commissure,  containing  blue-stained  fibres,  encloses 
the  central  canal  of  the  cord.  This  is  lined  by  columnar 
ciliated  cells,  the  attached  ends  of  which  are  tapered,  and 
branch  into  the  surrounding  central  gelatinous  substance.  On 
each  side  of  this  a  branch  of  the  anterior  spinal  artery  is 
frequently  seen  in  cross  section.  The  white  substance  is  com- 
posed of  medullated  nerve  fibres  embedded  in  a  spongework  of 
special  connective  tissue,  the  neuroglia.  This  forms  a  contin- 
uous layer  upon  the  surface  of  the  cord,  and  from  it  septa  pass 
inwards,  giving  off-sets  in  all  directions.  Nuclei  are  found  in  it. 
The  nerves  seen  in  cross  section  differ  much  in  size.  Forming 
the  centre  of  each  fibre  observe  the  axon  surrounded  by  the 
medullary  sheath,  which  frequently  exhibits  concentric  mark- 
ings, medullary  segmental  junctions. 

Fresh  cord,  for  nerve  fibres.  Place  a  small  fragment  of  the 
white  substance  upon  a  slide  with  a  little  normal  saline,  cover 
and  compress  slightly.  (H)  Note  the  medullated  fibres,  which 
soon  exhibit  regular  fusiform  expansions  as  the  medullary 
sheath  swells.     This  indicates  the  absence  of  the  neurilemma. 

Spinal  cord  of  cat.  (Injected  blue  gelatin  mass,  p.  Formol 
2p-c->  c.  G.,  m.  B.)  (Z)  Find  the  anterior  spinal  artery  entering 
through  the  ventral  fissure  and  breaking  up  into  branches  at 
the  commissure,  from  which  they  radiate  into  the  grey  sub- 
stance. A  superficial  set  of  vessels  are  distributed  upon  the 
surface  in  the  pia  mater,  the  branches  of  which  converge 
through  the  white  substance  to  the  grey,  which  they  do  not 
always  reach.  The  arteries  are  terminal.  (H)  Note  the  rich 
capillary  distribution  in  the  grey  matter. 


94  NERVOUS    SYSTEM. 

The  following  sections  of  the  human  spinal  cord  are  for  the  purpose 
of  studying  the  regional  differences  presented  by  it,  and  are  all  pre- 
pared in  the  same  way.     (p.    23,   s.   Eosin,  m.   B.) 

Conus  medullar  is.  (Z)  The  white  substance  forms  a  narrow 
margin  around  the  central  grey  matter.  The  horns  are 
rounded  and  the  commissure  is  broad.  The  lateral  portion  of 
the  ventral  horn  contains  a  number  of  large  nerve  cells.  The 
dorsal  root  is  broad  at  its  entrance  into  the  cord.  (H)  Find 
the  groups  of  cells  in  the  ventral  horn,  and  note  the  group 
previously  mentioned  (probably  a  limb  area,  Sherrington).  The 
cells  of  the  central  canal  are  usually  well  seen. 

Lumbar  cord.  The  whole  section  is  larger  than  the  last, 
and  there  is  much  more  white  substance.  The  ventral  horns 
are  much  more  bulbous  and  contain  many  large  nerve  cells, 
{H)  chiefly  in  its  ventral  and  lateral  portions.  The  dorsal 
horn  is  also  rounded  and  the  dorsal  nerve  root  sends  strands 
of  fibres  along  the  mesial  side  of  the  horn  to  enter  its  sub- 
stance nearer  the  neck.  The  central  canal  is  wider  trans- 
versely. 

Cervical  cord.  The  whole  section  is  larger  and  somewhat  more 
oval  transversely,  and  there  is  a  proportionately  greater  amount 
of  white  substance  in  the  posterior  columns.  The  grey  matter 
is  slimmer-looking  than  in  the  lumbar  region  though  its  mass 
is  about  the  same.  The  ventral  cornu  is  more  expanded  later- 
ally, the  angular  recess  between  the  ventral  and  lateral  horns 
being,  as  in  the  lumbar  region,  filled  up  with  cells— containing 
grey  matter.  In  the  upper  cervical  region  this  protuberance 
diminishes  and  the  lateral  cornu  becomes  prominent  through 
the  emergence  at  that  point  of  the  fibres  of  the  spinal  accessory 
nerve. 

Bulb.  T.S.  Man,  below  the  olive,  (p.,  &c,  the  same  as 
for   the  last.)      The  canal  is  still  central.     The   ventral  cornu 


BULB. CEREBELLUM.  95 

of  the  grey  matter  is  cut  off  from  the  remainder  by  the  decus- 
sation of  the  pyramids,  and  the  dorsal  cornu  are  twisted 
laterally.  Two  new  masses  of  grey  substance,  the  Gracile  and 
Cuneate  nuclei,  are  seen  in  the  corresponding  dorsal  columns. 
On  each  side  of  the  ventral  fissure  note  the  pyramids  from 
which  the  fibres  decussate  into  the  lateral  columns.  On  the 
dorso-lateral  part  of  the  section  note  the  crescentic  mass  of 
white  substance,  the  restiform  body.  The  fibres  of  the  twelfth 
nerve  may  be  seen  passing  from  the  ventral  cornu  and  running 
to  the  surface  on  the  outer  side  of  the  pyramid. 

Bulb.  T.S.  Man,  through  the  olive,  (p.  same  as  the  pre- 
ceding.) The  section  has  a  rounded  quadrangular  outline,  the 
dorsal  corners  being  formed  by  the  restiform  bodies,  the 
ventral  ones  by  the  olives,  which  are  rendered  prominent  by 
the  sinuous  streak  of  grey  substance  which  they  contain. 
Between  the  latter  on  each  side  of  the  ventral  fissure  are  the 
pyramids.  On  their  free  surface  note  the  external  arcuate 
fibres.  Dorsally  find  the  floor  of  the  fourth  ventricle,  against 
which  the  grey  matter  has  become  applied.  It  forms  two  or 
three  small  projections  ventral  wards  where  the  tenth  and 
twelfth  cranial  nerves  have  their  origin. 

Cerebellum.  Man.  T.S.  Convolutions,  (p.  the  same  as  the  27 
last.)  (L)  The  foliations  of  the  grey  matter  have  a  core  of  white 
substance  (stained  dark).  The  grey  substance  exhibits  two 
distinct  layers — the  outer,  molecular,  and  the  inner,  granula/r. 
At  the  junction  of  the  two  (H)  find  Purkinje's  cells  (not 
well   seen). 

Cerebellum.  Cat.  T.S.  Convolutions,  (p.  8,  s.  22  &  24, 
c.  P,  m.  B.)  (Z)  Recognise  the  granular  layer  which  is 
brought  out  prominently  by  the  haemalum  stain.  (H)  Examine 
the  Purkinje's  cells,   the   antler-shaped  processes  of    which    can 


96  NERVOUS    SYSTEM. 

be  traced  some  little  way  into  the  molecular  layer.  The 
spread  of  the  branches  is  in  a  plane  transversely  to  the  length 
of  the  convolutions. 

Pons  Varolii.  Man.  T.S.  (p.  23,  s.  Eosin,  m.  B.)  Its  mass 
is  divisible  into  a  larger  ventral  portion,  in  which  there 
are  large  transverse  bundles  of  fibres  interspersed  amongst 
the  cut  bundles  of  the  pyramidal  tracts.  The  dorsal  portion  is 
marked  off  below  by  the  fillet,  a  well  marked  angular  tract  of 
fibres.  Near  the  dorsal  surface  is  the  aqueduct  of  Fallopius, 
surrounded  by  grey  substance  (origin  of  the  fourth  nerve). 
Note  the  dorsal  eminences  formed  by  the  posterior  corpora 
quadrigemina. 

Midbrain,  crura  cerebri  and  corpora  quadrigemina.  Man. 
T.S.  (p.  the  same  as  the  last.)  The  tegmental  (dorsal)  portion 
contains  the  aqueduct  surrounded  by  a  thick  layer  of  grey 
matter  (origin  of  the  third  nerve),  and  shows  the  prominences 
of  the  corpora  quad.  The  crusta  (pes)  forms  the  crus  proper 
on  each  side,  and  consists  of  distinct  masses  separated  from 
the  tegmentum  by  the  substantia  nigra,  which  is  a  broad  band 
of  grey  matter.  The  crusta  is  composed  of  fibres  cut  trans- 
versely, the  middle  portion  of  which  is  the  continuation  of  the 
pyramidal  tract  (long  motor).  Above  the  substantia  nigra  in 
the  tegmentum,  and  placed  laterally,  is  the  fillet  (sensory  tract), 
whilst  near  the  middle  line  on  each  side  are  the  round  masses 
of  the  red  nucleus. 

Cerebral  cortex.  Man.  V.T.S.  Convolution,  (p.  the  same.) 
The  grey  matter  is  pink  and  the  medullary  is  blue,  and  consists 
of  axons  proceeding  to  and  from  the  grey  matter.  The  posi- 
tion  of  the  pyramidal   cells  can  be  made  out. 

Cerebrum.  Man.  V.T.S.  Ascending  frontal  or  parietal 
convolution,    (p.   Golgi,  27,  s.  24,  m.  B.,  uncovered.)     (L)  Find 


CEREBRAL  CORTEX. — NEUROGLIA.  97 

a  successfully  stained  pyramidal  cell.  Note  its  shape  and 
processes.  (//)  The  chief  dendritic  process  is  apical  which 
runs  to  the  periphery,  giving  off  short  lateral  branches  and 
terminating  fanwise  near  the  periphery.  Lateral  processes 
come  off  the  cell  body  itself.  A  thin  axon  courses  centrally, 
and  gives  off  collaterals.  The  cells  of  the  polymorphic  or 
fourth  layer  are  small  and  have  few  dendrites  and  a  fine 
peripheral  axon.  Fine  vertical  filaments  unconnected  with  cells 
are  also  to  be  noted. 

Neuroglia   cells   are    scattered    in    the    cortex,    the   body   is 
small  and  emits  numerous   delicate  branching  processes. 


C  ... 


CHAPTER    XVI. 
MALE    ORGANS    OF    GENERATION. 

Testis  and  epididymis.     Man.    (p.  11,  s.  22  &  24,  c.  P.,  m.  B.) 

The  testis  proper  is  enclosed  in  a  thick  fibrous  capsule  (tunica 
albuginea);  within  this  is  the  simple  tubular  structure  filled  with 
cells.  Dorsally  will  be  found  the  epididymis,  consisting  of  large 
tubules  separated  by  much  fibrous  tissue,  in  which  are  blood 
vessels.  (IT)  The  cells  in  the  tubules  can  be  studied  better 
in  the  next  preparation.  The  canals  of  the  epididymis  are 
surrounded  by  plain  muscle  and  contain  two  or  three  layers  of 
cells,  the  innermost  of  which  are  columnar  with  long  cilia. 

Testis  of  Rat,  for  spermatogenesis,  (p.  9,  s.  22  &  24,  c.  P.,  m.  B.) 
(H)  Find  T.S.  tubules  that  show  sustentacular  cells,  the  ends  of 
which  are  expanded  by  spermatoblasts  applied  to  their«free  ends; 
in  them  the  heads  of  the  spermatozoa  can  be  seen.  Note  the 
flagella  of  the  latter  projecting  into  the  lumen.  The  remaining 
cells  are  known  as  the  spermatogenic  cells,  which  vary  in  shape 
from  spheroidal  to  squamous,  and  exhibit  various  nuclear  figures. 
Between  the  tubules  in  the  lymph  spaces  occasional  narrow 
tapering  columns  of  interstitial  cells  are  met  with. 

Vas  deferens.  Dog.  (p.  11,  s.  22  &  24,  c.  P.,  m.  B.)  (L)  and 
(If)  An  external  coat  of  plain  muscle,  to  which  the  mucosal  sur- 
face, covered  by  ciliated  epithelium  like  that  of  the  epididymis, 
is  loosely  attached. 

Prostrate  gland.  Dog.  (p.  11,  s.  22  &  24,  c.  P.,  m.  B.)  (L) 
A  lobulated  true  gland  embedded  in  a  fibro-muscular  capsule. 
The  racemose  acina,  with  wide  lumen,  are  lined  by  small  cylindrical 
cells  with  distinct  nuclei.     (II)    The  cytoplasm  is  finely  granular. 


FEMALE  ORGANS  OF  GENERATION.  99 

The  short  ducts  are  furnished  by  the  same  kind  of  epithelium. 
The  urethra  membranous  portion,  lined  by  transitional  epithelium 
at  its  junction  with  the  neck  of  the  bladder,  is  frequently 
included  in  the  section. 

FEMALE  ORGANS  OF  GENERATION. 

Ovary.     Rabbit,  for  germinal  epithelium,     (p.  16,  s.  22  &  24, 

c.  P.,  m.  B.)     (L)  and  (//)    Upon  the  surface  find  the  single  layer 

of  small  ovoid  cells  resting  upon  the  stroma  of  the  organ.     In 

the  latter,  a  little  below  the  surface,  there  is  a  zone  of  young  ova. 

Originating  in  the  surface  layer  they    become    included  in  the 

stroma,   thereafter    increasing    in    size.     Large  ones  in  Graafian 

follicles   occur   further  in  and  tend   to   approach   the   surface  as 

they   enlarge.     Rounded   masses,  corresponding    to    the    former 

in  size,    or  larger,    and    filled   with    blood    or    cellular   contents, 

the  Corpora  lutea,  should  also  be  sought  for.     Find  the  broad 

ligament  or  attachment  of  the  ovary.     In  it  are  contained  the 

blood   vessels,    &c,    going    to    the    organ.     (H)     Examine    the 

epithelium ;    the    subjacent    stroma    has    numerous    oval    nuclei. 

Trace,  if  possible,  the  inclusion  of  the  germinal  cells  in  the  stroma 

and  the  formation  of  the  Graafian  follicle  around  them.     In  this 

the  ovum  is  at  first  surrounded  by  a  single  layer  of  cells  (discus 

proligerous);  then  by  several  layers,  amongst  which  a  cavity  is 

produced*  by  accumulation  of  fluid  (liquour  folliculi).     Observe 

the  thick  cell  wall  of  the  ovum  (zona  radiata)  and  the  appearance 

of  the  nucleus,  which  varies  greatly  with  its  maturation.     The 

corpus  luteum  will,  if  just  formed,  namely,  if  the  ovum  which  it 

contained  has  been  recently  evacuated,  be  full  of  blood,  in  which 

fibrin  filaments  may  be  seen  ;  or  its  margin  ma}r  be  occupied  by 

large   cells,   the    blood   clot    being  reduced  to  a  central  stellate 

mass,  or,  again,  the  latter  may  have  disappeared  and  cells  only 

be  present. 


100  FEMALE  ORGANS  OF  GENERATION. 

Ovary.  Rabbit,  for  corpus  luteum.  (p.  2  d,  c.  P.,  Weigert's 
fibrin  stain  m.  B.)  In  this  preparation  examine  the  details  already 
mentioned  and  the  elements  of  the  stroma. 

Fallopian  tubes.  Cat.  T.S.  (p.  3/inj.,  s.  22  &  24,  c.  P.,  m.  B.) 
(L)  and  (II)  Externally  a  muscular  coat  (plain),  to  which 
internally  a  folded  mucosa,  covered  with  ciliated  epithelium,  is 
loosely  attached. 

Uterus.  Rabbit  or  Cat.  T.S.  (p.  3,  inj.,  s.  22  &  24,  c  P.,  m.  B.) 
(L)  and  (H)  The  vascular  mucous  lining  is  closely  packed  with 
long  tubular  glands  (cubical  and  columnar  spith)  which  have 
duct-like  openings  upon  the  surface.  Externally  to  this  is  the 
circularly  disposed  plain  muscle  in  many  layers. 

Vagina.  T.S.  Cat.  (p.  3,  inj.,  c.  P.,  m.  B.)  (L)  and  (H) 
The  surface  epithelium  is  stratified  squamous  and  rests  upon 
connective  tissue,  in  which  small  papillary  elevations  are  per- 
ceptible. Mucous  glands  will  be  found  in  this  layer,  and 
externally  plain  muscle  mingled  with  connective  tissue. 

Mammary  gland.  Human,  non-lactating.  (p.  11,  s.  22  &  24, 
c.  P.,  m.  B.)  (L)  The  glandular  tissue  is  scanty  and  widely 
separated  by  rather  dense  connective  tissue.  The  ducts  are 
narrow. 

Mammary  gland.  Cat.  Lactating.  (p.  11,  s.  13  &  17,  c.  P.,  m.  B.) 
The  secreting  structure  occupies  the  whole  gland,  and  the  acini 
are  polygonal  through  distension  and  mutual  compression.  The 
epithelium  is  closely  packed  along  the  walls,  the  ends  of  the  cells 
projecting  irregularly  into  the  lumen.  Note  the  fat  stained  black 
in  the  cells  and  in  the  contents  of  the  lumen.  Find  the  milk 
ducts  and  their  expansions  in  the  nipple.  The  nipple  is  covered 
with  squamous  epithelium,  and  on  its  sides  small  sebaceous 
glands  occur.  Small  strands  of  non-striped  muscle  may  also  be 
made  out  in  the  connective  tissue. 


CHAPTER    XVII. 
SENSE    ORGANS. 

Olfactory  epithelium.  Guinea  pig.  (p.  3,  s.  11,  22  &  24,  e. 
P.,  m.  B.)  (L)  The  mucosa  of  the  olfactory  region  is  covered 
with  many  layers  of  cells,  fusiform  and  columnar  in  shape  and 
non-ciliated.  Beneath  this  note  the  glandular  masses  (Bowman's 
glands)  and  the  numerous  nerves  making  their  way  to  the 
surface.  (H)  In  very  thin  sections  it  is  possible  to  recognise 
the  two  kinds  of  cells.  The  olfactory  cells  are  thin,  with  a 
swelling  over  the  nucleus ;  the  end  which  reaches  the  surface  is 
provided  with  short  bristle-like  processes.  The  other  cells  are 
columnar  near  the  surface,  fusiform  and  irregular  further  in. 

Isolated  olfactory  cells,  (p.  An  exposure  of  the  brown 
membrane  of  the  turbinated  bone  of  a  rabbit  for  24  hours  to 
one-third  alcohol,  five  minutes  in  lpc-  osmic  acid,  stain  in  19  in 
bulk ;  dissociate  and  diffuse  in  glycerin  jelly  (after  Stirling). 
The  thin  olfactory  cells  can  easily  be  recognised  from  the 
columnar  supporting  cells.  The  latter  are  irregular  and  branched 
at  their  attached  extremities. 

Eye.  V.T.S.  Head  of  mammalian  foetus,  for  the  general 
structure  and  origin  of  its  parts,  (p.  16,  s.  22  &  24,  c.  P.,  m.  B.) 
(L)  Find  the  cornea,  the  part  nearest  the  cutaneous  surface, 
and  note  its  continuity  with  the  external  coat  or  sclerotic  of  the 
eye-ball.  Within  this,  in  front,  is  the  voluminous  lens,  the  fibrous 
nature  of  which  is  perceptible  even  with  a  low  power  :  note  their 
backward  trend.  On  each  side  of  the  lens  posteriorly  find  the 
ciliary  bodies,  or  what  represents  them  at  this  stage,  a  slight 
thickening,  which  trace  into  the  thin  choroid.     The  pigment  will 


102  SENSE    ORGANS. 

probably- not  yet  be  developed,  and  this  coat  may  be  difficult  of 
detection.  The  retina  comes  next  as  the  inner  covering  of  the 
eye,  and  may  exhibit  two  distinct  layers  continuous  with  each 
other  in  front  (line  where  the  infolding  has  occurred)  if  the  eye 
is  young  enough.  The  outer  thinner  portion,  probably  containing 
pigment,  will  form  later  the  pigmentary  layer  of  the  retina. 
Internally  to  this  is  the  retina  proper,  i.e.,  that  portion  which 
subsequently  is  alone  connected  with  the  optic  nerve.  Trace  the 
optic  nerve  into  connection  with  the  retina  and  note  its  central 
artery.  As  it  is  not  always  possible  to  ensure  a  foetus  of  the 
younger  age,  at  which  1he  simpler  evolution  of  the  eye-cup  from 
the  brain  vesicle  is  best  seen,  this  description  is  intended  to 
apply  equally  to  older  preparations. 

C orneo-sclerotic  junction.  Dog  or  Cat.  Y.S.  (p.  8,  s.  22  and  24, 
c.  P.,m.  B.)  {L)  and  (11)  Externally  the  cornea  is  covered  with 
stratified  (conjunctival)  epithelium,  the  innermost  cells  of  which 
are  columnar ;  beneath  this  is  the  corneal  substance  of  dense 
fibrous  tissue,  amongst  the  layers  of  which  spindle-shaped 
corneal  corpuscles  are  disseminated.  On  the  inner  surface 
there  is  a  homogenous  membrane — internal  elastic  lamina — 
covered  by  a  single  layer  of  flat  cells.  The  junction  of  cornea 
to  sclerotic  is  indicated  by  the  occurrence  of  the  pigment 
and  blood  vessels  of  the  sclerotic.  Internally  to  this  is  the 
attachment  (Lig.  annulare  bulbi)  of  the  choroid,  which  is  the 
pigmented  and  vascular  coat  of  the  eye.  The  choroid  has 
three  subdivisions  : — The  Iris  in  section  hangs  into  the  anterior 
chamber,  and  the  surface  turned  towards  the  corea  is  covered 
by  a  single  layer  of  densely  pigmented  cells.  Throughout  its 
substance  there  are  branched  cells  and  blood  vessels.  Towards 
the  free  edge  bundles  of  plain  muscle  are  added  (Sphincter). 
Beneath  the  epithelium  of  the  inner  surface  there  is  a  fibrous 
layer  free   of    pigment.      The  pectinate    ligament    is   the    frayed 


CORNEA. — LENS.  —  RETINA.  103 

edge  of  the  internal  elastic  lamina,  and  its  fibres  cross  the 
angle  of  junction  and  penetrate  into  the  iris.  The  ciliary 
portion  of  the  choroid  is  pervaded  by  branching  pigment  cells; 
its  inner  surface  is  covered  by  a  deeply  pigmented  layer,  upon 
which  rest  clear  columnar  cells ;  these  are  continued  to  the 
retina.  This  inner  layer  is  much  folded  and  a  thin  homo- 
geneous membrane  is  attached  to  its  surface  (suspensory 
ligament  to  choroid  and  inner  limiting  layer).  The  strands  of 
the  ciliary  muscle  (plain)  will  be  observed  radiating  outwards 
from  the  corneo-sclerotic  junction  into  the  ciliary  body.  The 
union  of  the  choroid  and  retina  occurs  further  back  ;  observe 
the  angular  bend  of  the  surface  at  the  ora  serrata. 

Cornea.  Cat.  V.  &  L.S.  (s.  Ran vier's  gold  method,  c.  P.,  m.  B.) 
(//)  Examine  these  successively  for  the  distribution  of  nerves, 
the  superficial  plexus  of  which  is  best  seen  and  lies  immediately 
beneath  the  conjunctival  epithelium.  In  the  L.S.,  besides  an 
irregularly  distributed  plexus  of  nerves,  observe  the  branching 
processes  of  the  corneal  corpuscles. 

Lens  fibres.  Cod's  eye.  (Boiled  in  water,  teased  in  the  same, 
stained  s.  19,  m.  F.)  The  band-like  fibres  seen  on  edge  in  clusters 
exhibit  ridges.  Seen  isolated  in  side  view  they  are  smooth,  with 
denticulated  margins  (whereby  they  interlock). 

Optic    papilla.     Dog.     Y.S.     (p.   11,   22   &  24,   c.    P.,   m.    B.)    £Q 

Find  the  entrance  of  the  optic  nerve  through  the  sclerotic 
(cribriform  lamina)  and  its  overflow  laterally  over  the  choroid, 
and  the  appearance  of  the  retina  immediately  outside.  A 
central  section  will  show  the  central  artery  of  the  retina  cut 
lengthways. 

Retina  of  Cat,  for  general  structure,  (p.  8,  s.  22  &  24,  c.  P., 
m.  B.)  The  inner  layer,  i.e.,  that  which  lies  nearest  the  vitreous 
humour,  is  smooth  and  bounded  by  the  inner  limiting  membrane, 


104  SENSE    ORGANS. 

from  which  spring  the  fibres  of  Miiller.  These  run  outwards 
(invisible  beyond  the  third  layer)  to  the  outer  limiting  layer,  and 
form  the  sustentacular  system  of  the  organ.  The  nervous 
elements  are  next  in  order  from  the  first  layer  outwards  (2) 
fibrous  layer  of  nerve  fibres,  (3)  Ganglionic  layer,  nerve  cells, 
(4)  inner  molecular  layer,  (5)  inner  nuclear,  (6)  outer  molecular, 
(7)  outer  nuclear,  (8)  outer  limiting,  (9)  rods  and  cones,  (10) 
pigmentary  layer.  Layers  (5)  and  (6)  are  stained  and  so  stand 
out  prominently.  (H)  Examine  the  different  layers  and 
observe  that  the  cones  are  much  less  numerous  than  the  rods. 

Retina,  Frog.  Illuminated,  (p.  8,  s.  17,  c.  P.,  m.  B.).  {L) 
Recognise  the  outer  or  pigmentary  layer,  also  the  two  nuclear 
layers  (stained  red),  and  the  outer  limiting  layer,  which  sepa- 
rates the  external  nuclear  from  the  layer  of  rods  and  cones. 
In  the  latter  the  rods  are  the  prominent  structures,  particu- 
larly on  account  of  their  large  outer  segments,  which  are 
unstained,  and  between  which  prolongations  from  the 'pigment 
cells  pass  nearly  to  the  limiting  layer.  The  external  part  of 
the  pigment  cell  contains  less  pigment  granules,  and  the 
nucleus  is   well   seen. 

Retina.,  Frog.  Killed  after  being  kept  12  hours  in  dark- 
ness, (p.  same  as  before.)  The  distribution  of  the  pigment 
is  the  point  of  interest.  Observe  that  it  is  retained  in  the 
body  of  the  cell  itself,  very  little  of  it  occurring  between  the 
rods.  It  is  now  possible  to  find  the  cones ;  these  are  small 
.spindle-shaped  structures,  which  are  placed  at  various  distances 
from  the  outer  limiting  layer,  with  which  they  remain  con- 
nected by  a  delicate  process,  and  often  project  some  way 
towards  the  pigmentary  layer. 

The  Ear.  Cochlea.  Guinea  pig.  L.S.  through  the  modiolus. 
The  bulla  being  exposed   and  opened,  the  cochlea  is   removed^ 


COCHLEA. — SEMI-CIRCULAR    CANALS.  105 

the  lower  turn  punctured,  (p.  9  for  one  hour,  transfer  to  half 
strength  14a  until  decalcified,  then  increasing  alcohols,  s.  22 
and  24,  c.  C,  clear  in  origanum  oil,  m.  B.)  (Z)  Find  the  coch- 
lear tube  in  section  and  the  osseous  spiral  lamina  which 
projects  into  it.  The  latter  has  two  lips  separated  by  the 
sulcus  spiralis.  From  the  longer  (tympanic)  lip,  the  basilar 
membrane  stretches  across  the  tube  to  be  attached  to  its 
outer  side  by  the  spiral  ligament.  Inwards  from  the  upper 
(vestibular)  lip  or  limbus  the  membrane  of  Reissner,  one  cell 
thick,  stretches  to  the  outer  wall,  where  it  becomes  continuous 
with  the  epithelium  of  the  spiral  ligament,  thus  enclosing  the 
triangular  cochlear  canal  of  the  membranous  labyrinth.  That 
portion  of  the  cochlear  tube  which  lies  outside  the  basilar 
membrane  is  the  scala  tympani  (to  fenestra  rotunda),  and 
that  outside  Reissner 's  membrane  is  the  scala  vestibuli  (to 
fenestra  ovalis).  The  lamina  spiralis  ossea  contains  the  spiral 
ganglion  and  nerves  passing  to  the  organ  of  Corti.  The  mem- 
brana  tectoria  attached  to  the  surface  of  the  limbus  is  fusiform 
in  section,  with  its  free  end  curled  up,  and  extends  over 
Corti's  organ.  (H)  The  organ  of  Corti,  with  its  inner  and 
outer  (rods)  sustentacular  cells  (enclosing  the  spiral  canal), 
reticular  membrane,  hair  and  Deiter's  cells,  then  Hensen's  cells 
on  the  external  side  forming  a  rounded  mass,  become  contin- 
uous with  the  flatter  epithelium  lining  the  remainder  of  the 
canal.  The  stria  vascularis,  a  thickening  of  the  membranous 
wall,  is  placed  between  the  spiral  ligament  and  the  attachment 
of  Reissner's  membrane.  In  this  preparation  a  section  of  the 
Eustachian  tube  is  often  included.  It  consists  of  an  incomplete 
tube  of  hyaline  cartilage,  lined  by  a  thin  mucosa,  with  a 
superficial   covering   of  columnar   ciliated   epithelium. 

Semi-circular  Canals.     L.S.     Ampulla,  Guinea  pig.      (p.  osmic 
1  pc-    4    hours,    then   14(a),  s.  22  &  24,  c.  P.,  m.   B.).      (L  &  H) 


106  SENSE    ORGANS. 

Find  the  ampullary  expansion  of  the  semi-circular  canal,  and 
recognise  that  it  is  lined  with  flat  epithelium,  lying  close  to 
the  osseous  walls.  On  the  outer  wall  will  be  found  the  crysta 
acoustica  in  T.S.  On  the  latter  the  lining  epithelium  forms 
a  cluster  of  larger  cells,  from  which  project  hair-like  processes 
into  the  cavity  of  the  tube.  Note  that  the  crysta  is  arranged 
with  its  length  across  the  axis  of  the  tube. 


APPENDIX    TO    THE    HISTOLOGICAL    SECTION. 

PRESERVING,    FIXING    AND    HARDENING    FLUIDS. 

General  Rules. —  /.  Fix  as  soon  after  death  as  possible.  2.  The  size 
of  the  piece  of  tissue  to  be  treated  is  of  the  first  importance  and  will  cur;/  with 
the  density  of  the  tissue  and  the  penetrating  power  of  the  reagent  used.  3.  Not 
less  than  20  volumes  of  fluid  to  one  of  the  tissue  are  to  be  used,  and  the  object 
should  be  suspended  by  a  thread  in  the  upper  part  of  the  reagent  or  laid  on 
a  bed  of  absorbent  cotton  so  that  the  fluid  may  have  ready  access. 

1.  Normal    saline    solution.      0.6  p-c-     NaCl     (6s    of     dry     NaCl 

are  dissolved  in  1,000 cc  of  distilled  water).  This  fluid  is  used  for 
the  examination  of  fresh  tissues,  and  delays  changes  in  them  for  a  short 
time;  though  not  perfectly  "indifferent,"  owing  to  its  convenience  is 
much  used.  Egg  white  is  a  useful  substitute.  Aqueous  humour  or 
iodised  serum  (blood-serum  to  which  iodine  crystals  have  been  added) 
are  sometimes  used. 

2.  Alcohol.     An   indispensable   reagent   in   histology. 

(a)  Absolute  alcohol  is  chiefly  used  for  final  dehydration  of  tissues. 
Used  alone  it  fixes  and  hardens  epithelial  structures  well.  24  hours  to 
several  days  according  to  the  size  and  density  of  the  tissue. 

(b)  One-third  alcohol  (Ranvier).  "Alcool  au  tiers."  1  part  90  pc 
alcohol  and  2  parts  water.  Ranvier's  original  receipt  is  "Alcool  a 
36°  de  Cartier  "  (88*5 pc-  pure  alcohol)  1  part,  water  2  parts.  Dissociates 
epithelial    structures   in   24   to   48   hours. 

(c)  After-hardening.  With  increasing  strengths,  starting  from  70 pc-, 
several  changes,  at  intervals  of  24  hours,  through  80p-c-  to  90p-c.  In 
the  latter,  tissues  or  sections  may  be  kept  stored  for  later  use  in 
well-stoppered  bottles.  Tissues  fixed  in  chromium  salts  should  either 
be  washed  in  water  before  the  alcohol  treatment,  or  be  kept  in  the  dark 
until  all  colour  has  been  washed  out  by  changes  of  alcohol,  70 pc-, 
otherwise  a  green  precipitate  is  apt  to  form.  After  corrosive  fixing, 
see  precautions  indicated  later.  Prolonged  after-hardening  in  strong 
alcohol  (methylated  spirit)  for  several  weeks  confers  great  firmness 
upon  tissues  and  enables  them  to  resist  the  strain  of  embedding 
more  perfectly. 

(d)  Methylated  spirit  does  well  for  nearly  all  purposes  as  strong 
alcohol.  If  methylated  spirit  loses  its  clearness,  and  turns  at  all 
milky  on  dilution  with  water,  it  is  unfit  for  use,  being  surcharged 
with   resins. 


108  APPENDIX    TO    THE    HISTOLOGICAL    SECTIOX. 

3.  Copposive  sublimate.  HgCL.  A  saturated  solution  in  water 
or  normal  saline.  Fixation  takes  place  rapidly  in  from  five  minutes  to 
two  hours,  the  time  being  determined  by  the  density  and  size  of  the 
piece  of  tissue.  As  this  reagent  does  not  penetrate  freely  small  pieces 
only  can  be  fixed.  They  must  not  exceed  6mm  thick,  and  these  will 
take  two  hours  to  fix.  Thinner  though  wider  pieces  will  take  a  shorter 
time. 

Injection  through  the  blood  vessels  is  the  best  method,  complete  penetra- 
tion being  secured  in  a  few  minutes.  The  parts  must  then  be  cut  up  rapidly 
under  running  water. 

After-treatment  with  increasing  strengths  of  alcohol  tinted  sherry 
colour  with  iodine  solution  until  discoloration  ceases.  The  Iodine 
helps  to  remove  uncombined  corrosive  sublimate  which  would  otherwise 
form  crystalline  deposits  and  obscure  the  sections.  Alcohol  aids  this 
removal,  as  corrosive  is  more  soluble  in  it  than  in  water  (33  parts  in 
100  in  alcohol,  25  in  ether,   7  in  water). 

4.  Alcoholic  copposive.  50 cc  of  70p-c-  alcohol,  50 cc  of  saturated 
solution  of  HgCl2  in  70p-c-  alcohol,  6  drops  of  HN03.  Only  very 
small  pieces  4mm  thick  can  be  fixed  in  this.  Time  required  1  to  4 
hours.      Then  increasing  strengths  of  alcohol. 

Nate. — Whilst  the  aqueous  solution  is  one  of  the  best  general  reagents  the  alcoholic 
form  is  especially  good  for  ganglia  and  glandular  tissues  such  as  liver,  kidney  and 
salivary  glands.    Tissues  stain  well  after  corrosive  treatment. 

5.  Chromic  acid.  Solutions  from  0'2  up  to  1  p-c-  in  water  have  been 
used.  This  reagent  is  a  bad  penetrant,  soon  turns  the  tissues  brittle, 
and  is  now  seldom  used  alone.  It  fixes  well.  The  following  mixtures 
are  used  chiefly  for  epithelial  and  nuclear  structures. 

6.  Chpom-aeetie  fluid  (Flemming).  Chromic  acid  0'25  p-c-,  acetic 
acid  0*1  p-c-,  in  water,  Very  small  pieces  of  tissue  take  about  two  days, 
then  increasing  alcohols  the  weaker  strengths  being  several  times 
renewed,  until  discoloration   by  the  escaping  chromic  acid  ceases. 

7.  Chromo-formic  fluid  (Rabl).  To  200  cc  of  an  aqueous  0*33  re- 
solution of  chromic  acid  add  four  or  five  drops  of  formic  acid.  Time 
and   after-treatment  the  same  as  6. 

8.  Chpomo-nitPic  fluid  (Perenyi).  To  3  parts  of  0*5  p-c-  chromic 
acid  add  4  parts  of  10p-c-  nitric  acid  and  lastly  3  parts  of  strong  alcohol. 
Time  4  to  8  hours,  followed  by  the  same  after-treatment  as  6. 

9.  Chromo-aceto-osmic  fluid  (Flemming's  fluid).  A  much  used 
cytological  reagent.  There  are  two  formula*,,  the  weak  one  is  the  more 
useful.     Chromic  acid  1  P-c-  25  parts,  osmic  acid  1  p-c-  10  parts,  ascetic  acid 


APPENDIX   TO   THE    HISTOLOGICAL   SECTION.  109 

1 ',c-    10  parts,  water  55   parts.     Small   pieces   take    from  2  to  24   hours, 
followed  by  thorough  washing  in  water  and  after-hardening  in  alcohol. 

10.  Platino-aeeto-osmic  fluid  (Hermann's  solution).  Platinic 
chloride  1'"  15  parts,  glacial  acetic  acid  1  part,  osmic  acid  2  ',c-  2  or  4 
parts.  This  fluid  does  not  produce  "artificial"  networks  in  protoplasm. 
Time   and   after-treatment  same  as  9. 

11.  Muller's  fluid.  Dissolve  25«  potassium  bichromate  and  10  & 
sodium  sulphate  in  one  litre  of  water.  Change  the  fluid  on  the  2nd, 
4th,  6th,  and  14th  days.  Six  to  eight  weeks  are  required  to  produce 
the  necessary  toughness.  This  is  one  of  the  best  penetrants  and  a 
reliable  reagent.  After-treatment  with  alcohols  in  the  dark  until  dis- 
coloration ceases. 

12.  Muller  and  spirit.  3  parts  of  Muller,  1  part  of  spirit  (methy- 
lated). Let  the  mixture  cool  before  use.  After  3  days,  change  to 
bichromate  of  ammonia  2  p-c-  solution.  Hardening  is  sufficient  in  about 
three  weeks.  The  after-treatment  is  the  same  as  for  Muller.  A  good 
general  reagent.  This  mixture  forms  the  first  step  in  Hamilton's  method 
for  hardening  large  masses  of  brain.  Keep  from  the  light  and  use 
fresh. 

13.  Osmic  acid.  This  reagent  is  an  instantaneous  fixer,  and  has 
been  regarded  as  devoid  of  any  distorting  effect  upon  tissues.  It  pene- 
trates so  slowly  that  only  very  small  pieces  of  tissue  can  be  treated. 
As  a  fixing  and  hardening  reagent  it  is  used  for  nerves  whose  medullary 
sheaths  it  is  desired  to  recognise.  1  p-c-  solution  in  water  24  to  48  hours, 
followed  by  strong  alcohol  if  sections  are  recpiired.  Cut  in  paraffin.  Its 
vapour  from  a  2i'c-  solution  has  been  employed  for  fixing  glandular 
tissue  (Langley).  It  is  mostly  used  in  conjunction  with  other  agents, 
or  upon  sections  of  already  fixed  and  hardened  tissues  to  stain  fat,  which 
it  turns  black  and  for  which  0o  to  1  p-c    solutions  are  used,  12  to  24  hours. 

14.  Bone-softening1  fluids. 

(a)  Chromic  acid  12-,  HXOs  25 cc,  water  to  1000 cc  for  decalcifying 
bones,  teeth,  and  hardening  the  soft  parts  at  the  same  time.  Followed 
by  alcohol  treatment  the  same  as  11  in  the  dark. 

(b)  Picric  and  nitric  (or  sulphuric)  acid.  The  former  saturated  in 
water  containing  2&c-  nitric  acid.  Chiefly  useful  for  embryonic  bones 
and  teeth.  As  soon  as  the  lime  salts  are  removed,  after-hardening  by 
increasing  alcohols  is  necessary.  The  picric  acid  should  be  completely 
removed  bv  the  changes  of  alcohol. 


110  APPENDIX     TO     THE     HISTOLOGICAL     SECTION. 

(c)  It  is  best  to  fix  and  harden  first  by  means  of  Miiller  or  corrosive 

and    subsequently   to    decalcify    with    2p-c-  HN03  or   lp-c-    HCL      After- 
harden  with  alcohols. 

15.  Sulpho-pierie  Acid  (Kleinenberg).  To  a  saturated  aqueous 
solution  of  picric  acid  add  2p-c-  H.2S04,  let  the  pp  settle  for  an  hour, 
filter,  and  add  three  parts  of  water.  The  pieces  must  not  be  large,, 
treat  from  3  to  6  hours,  followed  by  increasing  alcohols,  by  which  all 
colour  should  be  removed.  Tissues  stain  well  afterwards.  Good  for 
young  tissues. 

16.  (a)  Formol  (Formaline)  is  a  saturated  solution  of  formaldehyde  in 
water,  and  contains  40p-c-  of  the  gas.  This  reagent  penetrates  well 
and  hardens  central  nervous  tissue  rapidly.  A  10 pc-  solution  in 
water  (formol  1  part,  water  9  parts)  is  commonly  employed  for  the 
first  24  to  48  hours,  to  be  followed  by  5p-c-  solution  until  the  required 
density  is  acquired,  when  a  2^p-c-  solution  may  be  used  for  subsequent 
preservation.  Large  masses  of  tissue  are  soon  penetrated  and  may  be  cut 
by  freezing  in  gum  without  impregnation. 

(b)  Formol— Miiller  (Orth  quoted  by  Kahlden).  (1  part  formol, 
50  parts  Miiller.)  This  requires  to  be  fresh,  as  the  mixture  becomes 
turbid  and  deposits  (even  in  the  dark)  after  about  four  jlays.  For 
rapid  hardening  of  tissues  at  35°  C.  in  3  to  6  hours.  The  pieces  are  then 
dipped  into  gum,  frozen  and  cut  or  followed  by  thorough  washing  and 
after-treatment  with  alcohol. 

STAINING. 

17.  Borax  carmine  (Grenadier).  Carmine  3  £,  borax  4  s,  water 
100 cc.  Warm  moderately  until  the  carmine  is  dissolved.  When  cold  add 
an  equal  volume  of  70 pc-  alcohol ;  or  the  latter  may  be  omitted. 
A  good  bulk  stain,  though  rather  slow  in  action,  48  hours  to  a  week, 
or  more.  After-treat  in  acid  alcohol,  1  p  c-  HC1  in  70  p-c-  alcohol  for 
24  hours,  this  concentrates  the  stain  on  the  nuclei  and  gives  a 
redder   tint. 

18.  Alum  carmine.  Boil  1«  carmine  in  100 cc  of  5p-c  potash 
alum  for  20  minutes.  Does  not  overstain  and  is  not  alkaline.  The 
addition    of    10 pc-   glacial  acetic   acid   improves   its   penetrative  power. 

19.  Picrocarmine,  picrocarminate  of  ammonia  (Ranvier).  Car- 
mine dissolved  in  ammonia  is  poured  into  a  saturated  solution  of  picric 
acid  in  water  until  saturation  is  reached,  which  is  indicated  by  the 
appearance  of  a  precipitate.  Evaporate  the  mixture  to  one-fifth  its 
bulk,    in    a    drying    chamber.       When    cool,    filter,    and    evaporate    the 


APPENDIX    TO    THE    HISTOLOGICAL    SECTION.  Ill 

filtrate  to  dryness.  The  dried  residue  has  the  colour  of  red  ochre, 
and  should  dissolve  completely  in  distilled  water.  Use  a  1  p-c-  solu- 
tion  or   stronger  ;    add    thymol   to    keep    it. 

20.  Hematoxylin.  Kleinenbergr's  formula.  Stock  solutions. 
1.  Saturated  solution  of  calcium  chloride  in  TO1'0  alcohol  containing 
alum  in  excess  ;  filter  when  wanted.  2.  Saturated  solution  of  alum  in 
70 1>c-  alcohol.  3.  Saturated  solution  of  hematoxylin  crystals  in  abso- 
lute alcohol.  Add  1  part  of  No.  1  to  8  parts  of  No.  2,  then  a  few  drops 
of  No.  3  until  a  moderately  deep  purple  colour  results.  This  keeps 
almost  indefinitely  without  depositing.  The  reddish  colour  turns  to 
the  characteristic  violet  on  the  addition  of  water.  It  stains  rapidly, 
and  is  especially  useful  for  staining  on  the  slide.  Diluted  with  3  or 
5   volumes   of    No.    2   it   stains   well  in   bulk.       Small  pieces   of   tissue. 

21.  Heidenhain's  Hsematoxylin  stain.  A  bulk  stain.  1.  A  0*5 p-c- 
solution  of  hsematoxylin  crystals  in  distilled  water.  2.  A0'3pc-  solution 
of  neutral  chromate  of  potassium  in  water.  Small  pieces  of  glands 
hardened  in  absolute  alcohol  are  placed  in  Nos.  1  and  2  successively, 
in  each  for  12  to  24  hours,  are  then  washed  in  water,  treated  in  alcohol, 
and  cut  in  paraffin.     The  colour  is  steel  grey. 

22.  Haemalum.  Hsematein  can  be  purchased,  and  a  moderately 
deep-coloured  solution  is  made  in  a  10 p-c-  solution  of  alum  ;  or  dissolve 
hematoxylin  crystals  in  strong  ammonia,  dry  in  air,  and  make  a 
solution  as  above.  This  is  one  of  the  best  nuclear  bulk  stains. 
Requires  four  or  more  days,  according  to  the  density  and  size  of  the 
tissue  treated.  It  does  not  overstain.  Followed  by  increasing 
strengths  of  alcohol  to  the  last  of  them,  a  small  quantity  of  eosine 
can  be  added  to  give  a  ground  stain  before  passing  the  tissue  into 
cedar   oil   for  paraffin   embedding. 

23.  Weig*ert-Pal  method,  Bolton's  modification  (Jl.  Anat.  atad 
Physiol.,  vol.  xxxii.,  p.  264).     For  brain  and  spinal  cord. 

1.  Fixing  and  hardening.  The  tissue  as  fresh  as  possible  is  placed 
in  a  large  quantity  of  5  p-c-  formaldehyde  (1  formol  to  7  parts  of 
water).  Change  occasionally  for  six  weeks ;  may  remain  for  six 
months  or  longer.  The  tissue  may  be  cut  at  the  end  of  a  week,  but 
the  sections  have  a  slight  tendency  to  frill. 

2.  Cutting.  Freeze,  without  previous  soaking,  in  gum.  Keep  the 
sections  in  5  p-c-  formaldehyde  until  wanted. 

3.  Mordanting.  Sections  are  placed  in  one  of  the  following : — 
Ferric  ammonium  sulphate  (iron  alum)  2p-c-,  osmic  acid  1  p-c-  (for  other 
mordants    see    original    paper).      The    former    yields    ultimately    a    blue 


112  APPENDIX    TO    THE    HISTOLOGICAL    SECTION. 

violet  and  the  latter  a  black  colour.  If  treated  with  the  iron  alum 
they  remain  in  it  for  24  hours  at  the  ordinary  temperature,  whilst  in 
the  case  of  the  osmic  acid  they  remain  until  of  a  fawn  colour. 

4.  Staining.  After  mordanting  wash  in  water  and  stain  in 
Kultschitzki's  hematoxylin  (hematoxylin  Is  dissolved  in  a  little 
alcohol  added  to  100 cc  of  a  2p-c-  solution  of  acetic  acid). 

5.  Oxydizing.  Wash  in  water,  place  in  0'25pc- solution  of  potassium 
permanganate  for  five  minutes. 

6.  Decolourising.  Wash  in  water  and  place  in  Pal's  decolouriser 
(oxalic  acid  Is,  solution  of  sodium  sulphate  Is,  distilled  water  200 cc). 
In  this  the}"  remain  until  the  grey  matter  is  decolourised.  If  this  does 
not  occur  in  five  minutes  repeat  the  processes  5  and  6.  Finally  rinse 
in  water  and  pass  through  absolute  alcohol,  through  toluene,  or 
chloroform  to  xylol  (Bolton),  and  mount  in  balsam.  The  great 
advantage  is  that  No.  4  solution  keeps  a  considerable  time  as  compared 
with   Weigert's  formula. 

24.  Eosin.  A  strongly  tinted  or  saturated  solution  in  water  or 
alcohol  is  employed  according  to  the  purpose  for  which  it  is  required. 
Strong  aqueous  solution  for  staining  blood  films  or  sections  on  the  slide. 
The  alcoholic  solution  is  used  for  imparting  a  ground  stain  in  bulk  when 
dehydrating  before  clearing  for  paraffin  embedding. 

25.  Spiller's  purple  or  fuchsine  are  used  in  strong  aqueous 
solutions.     These  stain  elastic  fibres  selectively. 

26.  Methylene  blue  (S.  Meyer's).  0'5pc-  to  saturated  solution 
in  normal  saline  for  staining  nerve  terminations  in  tissues.  In  time  it 
also  colours  nuclei  and  the  cement  substance  between  epithelial  cells. 
Free  access  of  oxygen  is  required  during  the  process,  as  colourless 
leuco-products  are  otherwise  formed  in  the  tissues.  The  stain  is 
evanescent  and  must  be  fixed  when  at  its  best  stage.  A  saturated 
solution  of  ammonium  picrate  in  water  is  employed  to  fix  the  colour 
when  it  has  developed  to  the  required  extent.  Subsequent  preserva- 
tion is  accomplished  in  glycerin,  to  which  an  equal  volume  of 
ammonium  picrate   has   been  added.     This  is  Dogiel's  method. 

Bethe  recommends  Ehrlich's  subcutaneous  injections  of  successive 
doses  of  methylene  blue  in  strong  solution.  Successful  results  can  be 
obtained  by  staining  very  small  pieces  of  the  fresh  tissue  in  a  saturated 
solution,  examining  small  fragments  from  time  to  time  under  the 
microscope  to  ascertain  the  result,  and  then  fix  first  by  15  minutes9 
treatment  in  ammonium  picrate  saturated  solution  in  water,  and  then, 
after  Bethe,   transfer  to  a  mixture  containing   ammonium    molybdate    1 


APPENDIX    TO    THE    HISTOLOGICAL    SECTION.  113 

part,  distilled  water  10  parts,  chromic  acid  (2',r  solution)  10  parts, 
and  1  drop  of  hydrochloric  arid,  h  to  4  hours.  This  turns  the  blue 
into  an  insoluble  compound,  and  at  the  same  time  hardens  the  tissue. 
I  i,r  osmic  arid  solution  may  be  substituted  for  the  chromic  acid  ; 
fixation  then  takes  longer,  as  much  as  24  hours.  Tissues  are  then 
washed,  and  may  be  stained  in  bulk  in  alum  carmine,  and  are  cut  in 
paraffin  (avoiding  long  exposure  to  alcohol). 

27.  Gram's  method  of  staining  bacteria  and  nuclear  structures. 

1.  Stain  for  2  to  5  minutes  in  a  solution  of  methyl  violet  in 
2*5  v-c-  carbolic  acid  solution  in  water. 

2.  Transfer  the  preparation  for  1  to  1*5  minutes  into  a  solution  of 
iodine  1,  iodide  of  potassium  2,  water  300  parts.  In  this  they  turn 
black. 

3.  Differentiation  in  alcohol,  until  the  colour  has  disappeared  and 
the  preparation  turns  of  a  pale  grey  tint. 

4.  Mount  in  Canada  balsam. 

The  feature  of  this  process  lies  in  the  use  of  the  iodine  solution, 
which  transforms  the  previous  diffuse  stain  into  a  selective  one,  by 
acting  as  a  mordant. 

Bacteria,  nuclei  (partially),  especially  those  in  mitosis,  plasma  cells 
(Mastzellen),  the  horny  layer  of  the  epidermis  and  serous  epithelium. 

A  ground  stain  may  be  imparted  to  the  cytoplasm  of  cells  by  adding 
a  little  eosin  to  the  last  alcohol  used  in  differentiating. 

This  method  can  be  used  for  blood  films. 

28.  Weig-ert's  fibrin  stain.  Sections  of  tissue  hardened  in 
alcohol  treat  as  follows  : 

1.  Stain  5  to  10  minutes  in  saturated  solution  of  gentian  violet  in 
anilin  water  (anilin  oil  5 cc  shaken  up  with  water  100 cc  and  filtered 
until  clear). 

2.  Wash  in  0-6  p-c-  NaCl  solution. 

3.  Dry  on  the  slide  with  filter  paper. 

4.  Decolourise  for  2  to  3  minutes  in  a  solution  of  iodine  in  iodide 
of  potassium  (1  :  2  :  100). 

5.  Dry  with  filter  paper. 

6.  Decolourise  with  anilin  oil  1,  xylol  2  parts. 

7.  Remove  the  anilin-xylol  with  xylol. 

8.  Mount  in  Canada  balsam. 

29.  Beneke's  modification  of  the  above,  for  general  purposes. 
The  strength  of  the  anilin  decolourising  solution  is  diminished  by 
mixing  2  parts  of  the  latter  with  3  of  xylol. 


-c .... 


114  APPENDIX    TO    THE    HISTOLOGICAL    SECTION. 

By  means  of  this  it  is  possible  so  to  manage  the  stain  as  to  colour 
dividing  nuclei,  connective  tissue  fibres  (blue-violet  to  red-violet), 
elastic  fibres  (red),  fibriilas  of  bone  and  Sharpey's  fibres,  striated 
muscle  and  neuroglia  of  nervous  tissue. 

30.  Carbol-Fuchsine.  Fuchsine  1,  in  100  parts  of  a  5p-c-  solution 
of  phenol  in  water.     To  this  add  10 pc-  alcohol. 

31.  Lofflep's  alkaline  methylene  blue.  To  a  saturated  alco- 
holic solution  of  methylene  blue  30 cc  add  1 cc  of  a  1  p-c-  solution  of 
potassium  hydrate  and  100 cc  water. 

32.  Nitrate  of  silver.  For  staining  endothelial  outlines  a  0'2  p-c- 
or  weaker  solution  to  1  p-c-  in  distilled  water  is  used.  The  surface  to  be 
treated  is  extended  without  stretching  (pinned  out  on  a  cork  ring  with 
hedgehog  bristles),  is  quickly  rinsed  with  distilled  water,  and  then 
flooded  with  the  silver  solution  which  is  allowed  to  act  for  three  or  four 
minutes  or  longer,  according  to  the  depth  of  staining  required.  Both 
sides  of  a  membrane  may  be  stained.  Rinse  again  with  distilled  water, 
and  place  the  tissue  in  70p-c-  alcohol  and  expose  to  sunlight. 

For  demonstrating  cell-spaces  in  tendon  or  connective  tissue,  or 
Ranvier's  crosses  in  nerves,  exposure  for  20  minutes  to  a  1  p-c-  solution 
will  be  necessary  (the  time  depending  on  the  light).  Wash  in  water  as 
soon  as  the  staining  is  complete  and  pass  into  balsam.  For  blood- vessels : 
wash  out  the  blood-vessels  with  a  2  p-c-  solution  of  nitrate  of  soda,  follow 
this  with  an  injection  of  a  0"2p-c-  solution  of  the  silver  salt,  and  inject 
without  loss  of  time  either  70 pc-  alcohol  or  a  solution  of  gelatine  (10  of 
dry  best  gelatine  in  100  cc  of  distilled  water).      Expose  to  sunlight. 

Golgi's  chromate  of  silver  process,  for  central  nervous  system,  nerve 
terminations,  and  secretory  channels  in  glands.  Tissue  hardened  in 
Miiller's  fluid  does  well.  A  small  piece  of  this,  4 mm,  or  about  £  in. 
thick  is  placed  in  0*75  p-c-  solution  of  silver  nitrate  for  24  to  48  hours; 
sections  are  cut  by  hand  or  by  the  freezing  method  without  impreg- 
nating but  simple  immersion  in  the  gum  for  a  few  minutes  so  as  to 
surround  the  preparation  with  gum  on  the  plate  of  the  microtome. 
Mount  the  sections  uncovered  in  balsam  on  the  slide  or  on  cover- 
glasses,  and  in  the  latter  case  when  the  balsam  is  dry  invert  the 
preparation  on  to  a  slide  upon  three  feet  of  wax  or  paper,  and  fix 
to  the  slide  with  a  strip  of  gummed  paper  or  a  label  with  a  circular 
hole  cut  in  it.  Rapid  drying  of  the  balsam  is  necessary,  as  the 
chromate   of    silver   deposit   soon   turns   granular. 

Golgi's  rapid  method.  The  fresh  tissue  is  placed  in  the  following 
solution  for  three  or  four  days :  Potassium  bichromate  3  %,  1 pc>  osmic 
acid    30 cc,   distilled   water   100 cc.     For  each    piece  of    4  "^  cubed    30 cc 


APPENDIX    TO    THE    HISTOLOGICAL    SECTION.  115 

are  required.  The  best  temperature  is  20°  to  25°  C,  then  rinse  ill 
distilled  water  and  transfer  to  a  solution  of  nitrate  of  silver,  0'5  to 
0'7  pc-,  for  24  hours  to  several  days.  The  rapid  process  is  best  for 
young  structures,  before  the  meyline  sheath  has  developed.  (8.  R. 
Cajal  recommends  the  addition  of  formic  acid,  1  to  2  drops  to  300 cc  of 
the  silver  solution,  when  dealing  with  cerebellum  and  cerebral  cortex 
which  are  nearly  fully  grown.) 

Double  impregnation  method  (S.  R.  Cajal).  After  the  treatment 
already  mentioned,  blot  off  the  silver  solution  ;  the  pieces  of  tissue 
are  placed  in  bichromate  of  potassium  6  or  7  K,  water  100 cc,  osmic 
acid  1  r>c-.  30  to  35 cc  or  less.  In  this  they  remain  for  two  days. 
Blot  off  the  surface  fluid,  and  return  to  the  silver  solution  for 
24  hours. 

33.  Gold  chloride.  {Ranvier's  boiled  gold.  TraiU  technique,  page  826.) 
Gold  chloride  1  pc-  solution  in  distilled  water  4  parts,  formic  acid  1  part ; 
boil  and  cool.  Place  small  pieces  of  tissue  in  this  for  20  minutes 
or  longer.  Wash  and  transfer  to  formic  acid  diluted  with  four 
volumes  of  water,  and  keep  in  the  dark  for  24  hours.  Dissociate 
in   glycerin.      Good   for   end   plates   in   muscle. 

Lemon  juice  method.  Ranvier,  I.e.,  page  813.  Place  the  tissue  in 
freshly  expressed  juice  (filter  through  flannel).  Transfer  to  1  p-c-, 
5  to  10  minutes,  until  it  has  become  transparent.  Transfer  to  1  p-c- 
gold  chloride  for  20  minutes,  then  wash  in  distilled  water ;  reduce 
in  50  cc  distilled  water  with  2  drops  of  acetic  acid,  in  the  dark,  for 
24   hours. 

Tartaric  acid  method.  Stain  the  tissue  in  1  p-c-  gold  chloride  until 
it  is  penetrated,  one-half  to  two  hours,  for  cornea,  rinse  in  water ; 
reduce  in  nearly  saturated  solution  of  tartaric  acid  kept  at  50°  C. 
(embedding  bath)  until  a  greyish  violet  colour  is  produced  :  20  minutes 
to   one  hour. 

34.  Moppurgo's  method  for  isolating  muscle  fibres.  Virchow 
Archiv.,  Vol.   10,  p.  540. 

1.  Fix  in  salicylic  acid  2'5p-c-  in  alcohol,  renew  several  times,  after 
a  week  replace  by 

2.  Concentrated  solution  of  salicylic  acid  in  icater,  which  should 
gradually  replace  the  alcoholic  solution.  After  all  the  alcohol  has  been 
removed,  then 

3.  Envelope  the  pieces  in  cotton  wool,  boil  in  the  aq.  solution  for 
an  hour  in  a  water  bath,  and  let  the  tissue  remain  in  the  cooled  fluid 
for  two  weeks. 


116  APPENDIX    TO    THE    HISTOLOGICAL    SECTION. 

4.  After  this  period  the  muscles  may  be  removed  with  a  spoon. 
They  will  be  found  practically  free  of  fat  and  connective  tissue,  and 
only  loosehy  attached,  but  in  their  original  relationship.  They  can  now 
be  detached  with  a  spatula  from  the  bone,  and  be  isolated  by 
teasing  or  pressure.  The  fibres  are  coagulated  and  tough.  By  careful 
separation  in  dilute  glycerine  the  fibres  can  be  completely  isolated  and 
measured. 

Injection  of  blood  vessels  with  coloured  g-elatin  masses. 

As  this  is  an  operation  which  the  student  is  ordinarily  not  required 
to  perform  himself,  a  sketch  of  the  process  will  suffice.  Access  to  the 
blood  vascular  system  is  gained  by  exposing  the  heart  of  an  animal 
with  the  least  injury  to  the  surrounding  parts,  in  order  to  guard  against 
the  escape  of  injection  through  accidentally  injured  blood  vessels. 
The  apex  of  the  heart  is  cut  off  and  the  blood  allowed  to  escape. 
A  glass  cannula  or  nozzle  is  secured  in  the  aorta,  and  is  connected 
with  the  injecting  apparatus.  A  ligature  is  disposed  around  the  ven- 
tricles ready  for  closure  in  order  to  control  the  venous  outlet.  In 
the  case  of  a  single  organ  the  cannula  is  secured  in  the  chief  artery 
and  bull-nosed  artery  forceps  are  used  to  control  leakages  and  the 
venous  channels.  The  injection  apparatus  is  a  brass  syringe  or  an 
appliance  acting  under  continuous  air  pressure.  When  all  parts  of 
the  object  appear  injected  the  venous  outlet  is  closed,  the  pressure 
is  maintained  a  little  longer  and  the  aorta  is  clamped  to  confine  the 
injection.  During  the  injection  avoid  the  introduction  of  air  with  the 
fluid,  arrest  leakage  from  injured  blood  vessels,  and  use  the  lowest 
pressure  that  will  suffice  to  drive  the  fluid  through  the  capillaries 
the  occurrence  of  which  can  be  recognised  by  the  change  of  colour  in  the 
tongue,  nose,  eyes,  &c.  The  gelatine  mass  is  then  set  by  immersing 
the  object  in  cold  alcohol,  after  which  the  parts  required  are  cut  into 
suitable   pieces   and   hardened   in   spirit. 

Blue  gelatin  mass.  Take  25  parts  of  a  saturated  solution  of  soluble 
Berlin  blue  (Briicke's  blue),1  warm  it  and  to  it  add  slowly,  with 
constant  stirring,  1  part  of  the  best  French  gelatin,  which  has  been 
allowed  to  swell  in  distilled  water,  and  has  then  been  melted  by 
heating  in  the  water  which  it  has  imbibed.  When  thoroughly  incor- 
porated filter  the  mixture  through  flannel  wrung  out  of  hot  Mater  ;  it 
is  then  ready  for  use.  Injected  parts  gain  by  being  preserved  at 
first  in  Miiller's  fluid  or  2 p-?-  bichromate  of  ammonia  the  colour  of  the 
mass  is  thereby  greatly  improved  (Ranvier).  After  staining  in  bulk 
with  borax  carmine  cut  by  freezing  in  paraffin. 

1  Can  be  obtained  from  Dr.  Griibler,  through  Kauthack,  18,  Berner's  Street, 
London,  W. 


APPENDIX    TO    THE    HISTOLOGICAL    SECTION.  117 

Carmine  gelatin  mass.  Take  4  k  of  carmine  and  dissolve  it  in  the 
least  quantity  of  strong  ammonia  in  a  mortar,  let  it  nearly  dry  up, 
much  superfluous  ammonia  is  thus  got  rid  of,  and  rub  it  in  50 cc  of 
distilled  water.  When  completely  dissolved  filter  and  warm.  Place 
10s  of  clear  gelatin  cut  up  small  into  50  cc  of  distilled  water.  When 
the  gelatin  is  completely  swollen  up  heat  it  in  the  water-bath  until  it 
is  dissolved.  Add  the  gelatin  to  the  carmine  with  constant  stirring  until 
incorporated,  and  then  add  drop  by  drop  a  10  p-c-  solution  of  acetic  acid  until 
the  colour  of  the  whole  is  changed  to  a  brighter  red,  the  transition 
is  a  very  noticeable  one ;  the  mixture  should  have  a  distinct  odour 
of  acetic  acid  and  have  an  acid  reaction.  The  carmine  is  thrown 
out  of  solution,  but  no  precipitate  should  be  visible  under  the  highest 
power.     Strain  through  flannel  which  has  been  wrung  out  of  hot  water. 


PART    II. 
CHEMICAL    SECTION. 


Students  ape  required  to  bring1  the  following :— One  dozen 
test  tubes,  19 mm  (§  in.)  in  diameter;  one  packet  of  filter  papers,  10 cm 
(4  in.)  in  diameter;  two  beakers,  No.  2  size;  two  porcelain  capsules, 
No.    3  size;  three  glass  rods,    lScm   (7  in.)  long;    a  test  tube  brush. 

The  following  ape  provided  fop  the  student  in  his  loekep:— 

A  test  tube  stand  ;  tripod  ;  retort  and  burette  stand  ;  wire  gauze,  5  in. ; 
mug,  half-pint,  with  wires  over  the  mouth  ;  a  thermometer,  100°  C. ; 
Bunsen  burner. 


CHAPTER    XVIII. 

The   quantities  to   be  used  for  each   test  are  given  in  length   of 
column   in  a   test   tube. 

CARBOHYDRATES. 

Those  in  italics  occur  in  the  organs  or  secretions. 


Mono- 
Glucoses. 

(C6H1206) 

Dextrose.  (Glu- 
cose or  Grape 
Sugar). 

Galactose. 

Levulose. 

Gl  ycuronic  Ac. 


Di- 

Saccharoses . 

(C12H22On) 

rl  mol.  Dextrose. 
Saccharose  - 

1 1  mol.  Levulose. 

Maltose    ...   2  mols.  Dextrose. 

(\  mol.  Dextrose. 

1 1  mol.  Galactose. 


Lactose 


Poly-saccharides 

Amyloses. 

(C6H10O6)n 

Starch. 

Soluble  Starch. 
Dextrins. 
Animal  Gum. 
Glycogen. 
Cellulose. 


(occ.  as  Gly-ates). 

All  optically  active  are  dextrorotary  excepting  Levulose. 


I.— GLUCOSES. 

Dextrose.  Dissolve  a  large  pinch  of  common  grape  sugar  in 
a    tube    full    of    warm    water,    and    test    as    follows : — 

1.  Trammer's  test.  To  4 cra  of  the  solution  add  2  drops  CuS04 
solution,  then  NaOH  solution,  until  the  hydrated  oxide 
of  Copper,  which  falls  at  first,  is  redissolved,  giving  a 
a  clear  blue  colour.  Boil  =  a  yellow  pp  of  suboxide  of 
copper    forms  =  Reduction.      (This    test    is    performed    in 


122  CARBOHYDRATES. 

the  same  way  as  the  Biuret  reaction  for  Proteids.  In 
the  latter,  however,  there  is  no  boiling.) 

Perform   a    control    test    with   water.       The    hydrated 
oxide  is   not  redissolved. 

2.  Fehling's   solution.     Take  4 cm,  boil   it,    then   pour  down  the 

side  of  the  sloped  tube  a  few  drops  of  the  sugar  solution. 
If  enough  sugar  is  present  an  orange  top  stratum  of 
suboxide  will  form  in  a  few  moments.  If  not  heat  again. 
Take  1 crn  of  the  reduced  fluid  and  add  NH3  until  it 
is  redissolved.     Note  the  volumes  required  to  do  so  (Pavy). 

3.  Add  one-quarter  volume  saturated  solution  Picric  Acid  to  4cm 

of  the  fluid,  and  then  a  few  drops  NaOH.  Heat  =  a  rich 
red  port  colour  results. 

4.  Moore1  s  test.     To  2  cm  of  the  fluid  add  an   equal   quantity  of 

]STaOH  solution,  boil  ==  a  yellow  to  deep  brown  colour 
results,  depending  upon  the  amount  of  sugar  present. 
There  is  an  odour  of  caramel,  especially  on  adding  weak 
H2S04. 

5.  Phenyl-hydrazin    test.     To  a   tube   three-quarters  full   of    the 

solution  add  one  knife-point  of  phenyl-hydrazin  and  one 
of  sodium  acetate.  Boil  in  the  water-bath  for  thirty  minutes 
or  more.  On  cooling,  or  before,  a  yellow  crystalline  or 
amorphous  pp  of  phenyl-glucosazone  separates.  Crystals 
fine  yellow  needles  in  feathery  clusters.  Examine  them 
under  the  microscope  (//). 

6.  Barfoed's  Reagent.     Performed  in  the  same  way  as  Fehling's 

test  =  reduction.  Herein  differs  from  milk  sugar,  maltose 
and  dextrin,   which  do  not  reduce  this  reagent. 

7.  Fermentation    test     with    yeast,    see    abnormal    urine    later. 


SACCHAROSE. — MALTOSE. — LACTOSE.  1  23 

II.— SACCHAROSES. 

CANE    SUGAR    GROUP. 

Cane  Sugar.     Apply  the  following  tests  to  a  solution  of   2  cm 
crystals   in   a   tube  full   of  water. 

1.  Solutions  do  not  reduce  (Fehling,  Trommer). 

2.  Easily   inverted  =  Heat  with  dilute    H2S04  to    100°    C,   very 

soon  a  reducing  sugar  is  produced  : — 

C12H22On  +  H20  =  C6H1206  (dextrose)  +  C6H1206  (levulose). 

Levulose  is  the  more  strongly  levorotary,  therefore  the 
mixture  exhibits  left-hand  rotation  to  the  extent  of  the 
difference  between  the  two. 

3.  BarfoeoVs  Reagent  is  not  reduced  by  cane  sugar. 
Maltose  is  a  reducing  sugar. 

Differs  from  dextrose  because  rotary  power  nearly  three  times  as  great — maltose, 
150° ;  dextrose,  56°  (10  p-c.  solution  at  20°).  Its  reducing  power  is  one-third  less. 
60  parts  dextrose  reduce  as  much  as  100  parts  maltose.  Maltose  can  be  trans- 
formed into  dextrose  easily  by  acids  and  ferments,  but  dextrose  not  into  maltose. 
Maltose  must  first  be  transformed  into  dextrose  before  its  absorption  into  the 
blood.     One   molecule   of    maltose    decomposes    into    two    molecules    of    dextrose. 

1.  BarfoeoVs  Reagent  is   not  reduced. 

2.  Phenyl-hydrazin    test,    see    dextrose.      Requires   several  hours 

boiling.  Yields  fine  yellow  needles  of  phenyl-maltosazone, 
shorter,  but  in  well-shaped  crystals  thicker  than  those  of 
phenyl-glucosazone. 

Lactose    is  a  reducing  sugar. 

Less  soluble  in  H20  than  dextrose.  Rotary  power  same  as  dextrose.  Insoluble 
in  alcohol.  Non-fermentable  by  yeast.  Lactose  must  be  transformed  into  dextrose 
before  it  can  be  assimilated.     If  injected  into  the  veins  it  appears  in  the  urine. 

1.  BarfoeoVs  Reagent  is  not  reduced. 

2.  Phenyl-hydrazin    reaction.       See  Dextrose.      Requires    longer 

boilino-.  Yields  fine  and  shorter  needles  than  the  other 
two   sugars   mentioned,   usually  in   heavy  clusters. 


124  CARBOHYDRATES. 

III.— AMYLOSES. 

Starch  Group. 

Starch.     Occurs  in  nature  as  granules  consisting  of  granulose 
in  a  skeleton  of  cellulose. 

1.  To   a    tube   two-thirds    full   of   cold  water   add   a   couple    of 

pinches  of  starch,  shake  briskly,  it  does  not  dissolve. 
Boil,  a  dull  solution  results.  If  enough  starch  is 
present  it  forms  a  gelatinous  mass  on  cooling.  Starch 
mucilage. 

2.  To   some   of  the   solution    add    iodine    solution  =  Blue  colour. 

Iodide  of  starch.  Heat  the  blue  solution,  the  colour 
disappears    to    return    on    cooling. 

3.  To  a  little   (1)  diluted  add  a  few    drops  of  NaOH.     Iodine 

does   not   give   a   blue   colour   in   an   alkaline   reaction. 

Transformations  of  Starch.  Take  as  much  powdered  starch 
as  can  be  heaped  on  a  penny,  place  it  in  a  beaker  and  rub 
it  into  a  cream  with  a  little  cold  water.  Add  this  gradually 
to  50 cc  water  which  is  boiling  briskly  in  a  beaker  and  stir 
thoroughly  until  all  the  starch  is  swollen,  which  will  take  from 
five  to   six  minutes.       Thick  starch  mucilage  (A). 

Place  a  little  of   this   in  a  tube.      Dilute   it   and  test 

with   iodine  and  with    Fehling.       No   reduction — absence 

of   sugar. 

As  soon   as   the   temperature   of  the  original  starch  mucilage 

has  fallen  to  40°  C.  it  thickens  markedly,  then  add  to  it  5 cc  of 

pancreatic    extract,  or    of    your    own    saliva1,   stir    continually. 

In   a   few   minutes   the   mucilage  will   turn  quite  fluid   through 

the  action   of  the  ferment. 

1  Obtain  your  own  saliva  as  follows  :— Hold  a  tube  against  the  lower  lip  to  catch 
the  saliva.  Open  your  mouth  slightly  and  breathe  the  vapour  of  acetic  acid  from 
the  reaK'nt  bottle.     At  least  10 cc  should  be  collected. 


piGESTION. —  ACTION    OF    SALTS.  125 

Take  half  a  tubeful,  boil  it  at  once,  and  when  cool 
dilute  some  of  it  and  test  with  iodine,  blue  iodide,  and 
with    Fehling — no    reduction.     Soluble  starch  (B). 

Make  two-thirds  of  a  beakerful  of  a  fresh  digestion  with 
cleared  mucilage  provided  for  you,  and  which  has  been 
obtained  by  allowing  some  more  dilute  mucilage  to  settle  until 
the  uns  welled  grains  and  non-carbohydrate  materials  have 
deposited. 

Prepare  several  tubes  of  weak  iodine  solution,  by  adding  a 
few  drops  of  the  solution  to  a  half  tube  of  water  in  each  case. 

Test  the  digestion  at  frequent  intervals  by  carrying  a  drop 
of  it  with  the  thermometer  into  one  of  the  tubes  containing 
iodine.  Take  specimens  as  follows,  boiling  each  at  once  to 
arrest   further   ferment   action  : — 

When  iodine  gives  a  red  port  colour — erythrodextrin  (C). 

When   iodine    ceases   to   give    this  colour   and    Fehling 
is   not   reduced — achrodextrin  (D). 

When  Fehling  gives  a  reaction — maltose  (E). 

Arrange  the  specimens  behind  their  corresponding  iodine 
reactions  in  the  tube  stand  ready  for  the  next  process. 

Precipitation  of  starch  and  its  derivatives  by  neutral 
salts.  Form  the  iodides  or  use  those  already  obtained  and 
saturate  some  of  each  with  Am2S04,  or  MgS04.  A  pp  results. 
The  pp  occurs  without  the  iodine,  but  more  slowly.  NaCl  is 
inactive. 

The  crystalline  carbohydrates  dextrose,  levulose,  cane  sugar. 
Lactose  and  maltose  do  not  yield  this  pp.  (R.  A.  Young, 
Jl.  Physiology,  Camb.   and  Lond.,  Vol.  xxii.,  pg.  405.) 

Commercial  dextrin  (British  gum,  made  by  heating  starch 
to   200°  C). 


126  CARBOHYDRATES. 

Dissolve  some  of  the  fawn-coloured  powder  in  water  (note 
the  smell). 

Test   with    iodine   and    Barfoed's  reagent. 
Saturate  a  small  quantity  of  the  solution  or  the  iodide 
with  Am2S04. 

Basic   lead  acetate  gives   no  pp. 

Glycogen  (animal  starch).  A  sufficiently  pure  aqueous 
solution  is  obtained  by  killing  a  rabbit  which  has  been  fed 
three  hours  before  on  carrots.  The  liver  is  at  once  removed, 
chopped  fine,  and  thrown  into  actively  boiling  water,  where 
it  remains  ten  minutes.  Proteids  are  removed  by  acidulating 
slightly  with  acetic  acid  and  boiling  two  minutes  longer.  The 
fluid  is  then  strained  through  mull  muslin,  cooled,  and  neutral- 
ised with  sodium  carbonate.  It  should  not  reduce  Fehling. 
Note  how  much  if  any  reduction  occurs.  Examine  the#  solution 
provided  for  you  as  follows  : — 

1.  The  solution  of  glycogen  is  markedly  opalescent. 

2.  Add  weak  iodine  to  a  portion — red  port  colour.     Effect  of 

heating  and   cooling  1 

3.  Saturate  some  or   the   iodide   with  Amj304  a   flocculent  pp 

results. 

4.  Boil    some   in   a   tube   with    dilute    H2S04  (0-25pc).      Test 

with   Fehling.      Reduction   indicates    the  formation  of  a 
reducing  sugar   (dextrose). 

5.  Barfoed's   reagent  is  not  reduced. 

6.  Basic  lead  acetate  gives  a  pp. 

The  lead  acetate  must  be  basic.     To  ensure  this  plumbic  acetate  is  boiled  with 
litharge  for  ten  minutes,  the  nitrate  will  be  basic  lead  acetate. 


CHAPTER    XIX. 

FATS. 

Saponification. 

1.  Take  a  little  melted  tallow,  add  2  volumes  of  10 pc-  ISTaOH, 

boil  with  constant  agitation  for  five  to  ten  minutes  until 
the  quantity  of  melted  fat  which  comes  to  the  surface 
when  the  tube  is  held  at  rest  is  much  diminished. 
Add  water,  boil  again,  cool,  run  through  a  wet  filter. 
The  nitrate  contains  the  soap  which  has  been  formed  = 
saponification. 

2.  Neutralise  8 cc  of  the  filtrate,  warm,  saturate  with  NaCl,  the 

soap  will  fall  in  flocculi ;  slight  warmth  assists  this 
separation. 

Emulsification . 

3.  Place  in  one  tube   (a)    some  soap   solution,  and  in   another 

(b)  the  same  quantity  of  water.  To  each  add  one-third 
volume  of  fresh  neutral  (litmus  paper)  olive  oil  and 
shake  them  briskly,  place  them  side  by  side  in  the  rack 
to  stand  for  15  minutes  :  note  the  difference  in  the  two 
emulsions.  Tube  a  will  present  a  uniforn  and  creamy 
emulsion. 

4.  To    some    cod    liver    oil    add    1    volume    carbonate    of    soda 

solution.  Shake  briskly,  an  emulsion  results.  Soap  is 
formed  by  the  union  of  the  fatty  acid  in  the  oil  with 
the  alkali. 


128  FATS. 

5.  To  a  solution  of  egg  albumin  add  1  volume  of  olive  oil. 
Shake,  rest,  a  more  or  less  perfect  emulsion  results, 
depending  on  the  strength  of  the  albumen  solution. 
The  mechanically  separated  fat  is  kept  suspended  by 
the  viscidity  of  the  proteid. 

Acetone  (dimethyl  ketone)  belongs  to  the  acetic  acid  series. 
Perform  the  following  test  on  a  2pc-  solution  of  commercial 
acetone  in   water.      Note  its  ethereal  odour. 

LegaVs  test.  A  few  drops  of  an  aqueous  solution  of  sodium  nitro- 
prusside  +  KOH  =  red  colour  which  rapidly  disappears, 
and  gives  purple  or  violet  red  with  acetic  acid.  See 
Creatinin. 

Interest  attaches  to  this  substance  owing  to  its  appearance  in  the  blood  and 
urine  in  diabetes  mellitus.  It  may  occur  temporarily  in  the  breath,  &c,  with  highly 
nitrogenous  diet. 


CHAPTER    XX. 
PROTBIDS. 

Albumins  and  Globulins.     Native  proteids. 

Egg  white.  (S.G.  1,045,  Alkaline,  contains  10p-c-  Proteids,  one- 
twentieth  of  which  is  globulin,   and  nearly  l  p-c-   salts. ) 

Break  an  egg,  decant  the  white  from  the  yolk  into  a  porcelain 
capsule,  cut  into  it  repeatedly  with  scissors  to  break  up  the  membranes, 
strain  through  wet  linen. 

Make  the  following  solutions  : 

1.  Measure  5CC  into  a  beaker  and  add  water  to  50 cc,  mix  thoroughly 
by  stirring.  The  turbidity  which  results  is  due  to  the  globulin  which 
is  thrown  out  of  solution  by  the  dilution  of  its  saline  solvent.  Strain 
through  mull  muslin  =  solution  egg  albumin. 

2.  To  another  solution  of  the  same  strength,  unstrained,  gradually 
add  with  constant  stirring  small  quantities  of  a  10  p-c-  solution  of  common 
salt  until  solution  of  the  globulin  is  effected.  Note  the  approximate 
percentage  of  salt  required  to  effect  this  solution.  Strain  =  solution  egg 
albumin  and  globulin. 

Utilise  both  of  these  solutions  to  perform  the  following  more  char- 
acteristic reactions,  taking  about  8CC  for  each  test. 

Colour  reactions. 

1.  Xanthoproteic  reaction.  Add  2  drops  of  HN03  a  pp  forms, 
heat  increases  it  and  produces  a  yellowish  tint.  Cool, 
add  NH3  an  orange  colour  results,  which  may  constitute 
the  whole  change  if  the  quantity  of  proteid  present  is 
insufficient  to  give  a  pp  with  acids  or  heat.  (Due  to 
nitro-derivatives,   Salkowski. ) 


130  PROTEIDS. 

2.  Millon's  reaction.      Add   5   or   6   drops   of  the  reagent  (acid 

nitrate  of  mercury)  a  white  pp  occurs  which  heating 
increases,  and  ultimately  turns  to  a  dull  brick  red.  In 
weak  solutions  a  red  colour  may  be  the  only  token  of 
a  reaction.      (Tyrosine,   Kiihne.) 

3.  Piotrowski 's   reaction.       Add     2    drops     of     CuS04    solution, 

then  sufficient  KOH  or  NaOH  solution,  until  the  white 
pp  occasioned  by  the  metallic  salt  is  redissolved  in  the 
organic  solution  and  a  transparent  bluish-violet  colora- 
tion is  produced.  Perform  a  control  test  with  water 
and  observe  that  the  pp  of  hydrated  oxide  of  copper 
which  takes  place  upon  the  addition  of  the  alkali  does 
not  dissolve.  This  test  is  also  known  as  the  Biuret 
reaction,  because  of  its  resemblance  to  that  obtained  with 
urea. 

Reaction  with  mineral  acids.      (H2N03,  H2S04,  HC1.) 

4  Add  2  drops  of  HN03  a  white  pp  results.  Or  by  the 
contact  method  (Heller's  test)  pour  HlSTOg  into  the  tube 
to  a  depth  of  2cm,  incline  it  and  flow  an  equal  quantity 
of  the  fluid  quietly  upon  the  acid,  a  cloud  will  form  at 
their  junction.  If  the  solution  be  poor  in  proteid  the 
cloud  will  disappear  on  agitating  the  fluids  together. 

Effects  of  metallic  salts,  &e. 

5.  Add  2  drops  of   acetic  acid,  then   2CC  or  3CC  of  ferrocyanide 

of  potassium,  there  is  a  white  pp. 

6.  Add  2  drops  of  acetic  acid  and  one-third  volume  of  saturated 

solution  of  picric  acid;    a  white  pp  results. 

7.  Mercuric    chloride.       A    few    drops    produce    a    white    pp. 

A  number  of  other  metallic  salts  do  the  same. 


ACTION    OF    REAGENTS.  131 

Miscellaneous  reagents. 

8.  Absolute   alcohol.      Take    5CC  of    the    solution,    add    an    equal 

volume  of  alcohol,  the  proteid  partly  falls  out  of  solution, 
not  however  completely. 

9.  Coagulation  by  Ether.       Upon   2  cm  in  a  tube  pour  an  equal 

quantity  of  ether,  a  cloud  forms  at  the  junction.  The 
fluid   must   be   neutral. 

10.  Tannin.     To   5CC  add   an   equal  volume   of   a    10  pc-  solution 

of  tannic  acid ;  a  white  pp  occurs. 

Precipitation   by    neutral    salts.       MgS04,    (NH4)2S04     written 
Am2S04. 

11.  Precipitation  of  globulins.     Half   saturate   5CC  of   No.   2   by 

adding  an  equal  volume  of  saturated  solution  of  Am2S04. 
Make  the  latter  by  dissolving  in  5CC  of  hot  water  as 
much  of  the  salt  as  will  dissolve,  and  whilst  warm  (40°  C) 
add  it  to  the  warmed  proteid  solution.  The  white  pp 
will  not  be  a  pronounced  one  owing  to  the  small  quantity 
of  globulin  present.  Complete  saturation  with  MgS04 
has  the  same  result. 

12.  Precipitation  of  dlbumin.    Saturate  completely  with  Am2S04. 

To  ensure  saturation  warm  the  fluid  to  about  40°  C. 
and  add  the  salt  until  some  of  it  remains  undissolved 
at  the  bottom  of  the  tube.  As  water  takes  up  about 
1  volume  of  the  salt  not  more  than  8CC  of  the  solution 
should  be  employed,  in  order  that  the  process  of  satura- 
tion may  not  be  too  protracted.  The  cloud  will  be  best 
seen  above  the  undissolved  salt. 


H  ft. 


132  PROTEIDS. 

Coagulation  by  heat. 

13.  Test  both  solutions  with  litmus  paper  and  acidify  both 
with  dilute  acetic  acid  (commercial  acid  of  33 pc  to  16 
volumes  of  water).  Heat,  it  is  not  necessary  to  boil, 
and  note  the  formation  of  a  coagulum,  the  density  of 
which   will   vary  with   the   quantity   of  proteid  present. 

Determination  of  the  temperature  of  coagulation. 

Use  a  ^-pint  tin  mug  as  a  water   bath,  with  wires  across  its  mouth 
to  support  the  tubes.     Fill  nearly  with  water  and  introduce  a  thermometer. 

Prepare    3    tubes   with    10cc  of  the   solution   in    each    tinted 
with  litmus  : 

(a)  The  original  solution  rendered  neutral  by  cautious  addition 

of  dilute  acetic  acid. 

(b)  Made  acid  with  dilute  acetic  acid. 

(c)  Made  faintly  alkaline  by  adding  small  quantities  of  carbonate 

of  soda  (4pc)  solution. 

Label  each  on  a  piece  of  paper  placed  in  the  mouth 
of  the  tube.  Raise  the  temperature  of  the  fluids  slowly, 
and  as  soon  as  40°  C.  is  reached  watch  carefully  for  any 
change  in  the  transparency  and  note  when  opalescence 
occurs,  then  solidification,  and  later,  at  a  higher  tem- 
perature, coagulation,  i.e.,  separation  of  flocculi.  Repeat 
with  solution  No.  2,  and  observe  that,  owing  to  the 
presence  of  the  globulin,  coagulation  commences  at 
a  lower  temperature. 

Globulins  alone. 

Myosin.     Fresh   meat   is   cleared   of   fat   and   tendons   and   is   finely 
chopped   up   and  washed  in  running  water  until  free  of  colour.      It  is 


GLOBULIN. — ALBUMINATES.  133 

then  extracted  with  10  r>c-  chloride  of  sodium  for  24  to  48  hours  in 
a  cool  place,  is  strained  through  muslin,  and  a  second  time  through 
a  plug  of  tow  placed  at  the  bottom  of  a  funnel. 

A  globulin  may  be  obtained  by  the  same  method  from  pea  meal, 
but  the  reactions  are  not  so  pronounced. 

1.  The  solution  of  myosin  is  slightly  opalescent. 

2.  Perform    the    reactions   for   proteids   and    note  carefully  any 

difference   in   the   reactions  where   any  occur. 

3.  Saturate    7  cc  of    the  solution   in  a  tube  with    MgS04,  filter 

into  another  tube  and  test  the  nitrate  for  the  presence 
of  a  coagulable  proteid,  by  acidification  and  boiling, 
there   will  probably  be  none. 

4.  Pour  some  of  the   solution   by  drops  into  a  tube  nearly  full 

of  water.  A  cloud  will  form  in  the  track  of  each. 
Explain   how   this  occurs. 

Albuminates  or  Derived  albumins. 

Alkali  albumin.  Form  alkali  albumin  by  adding  drops  of  caustic 
alkali  solution  (NaOH  or  KOH)  to  5 cm  of  pure  egg  white  in  a  tube. 
The  previously  fluid  albumin  will  soon  turn  into  a  clear  jelly,  adhering 
to  the  tube  when  inverted.  Fill  the  tube  with  water  and  stir  up  the 
jelly  with  a  glass  rod  until  most  of  it  is  dissolved. 

1.  Alkali     albumin     is     soluble     in     a     weak     alkali     but     is 

precipitated  on  neutralisation.  Add  litmus  solution  to 
a  distinct  tint  and  neutralise  with  dilute  acetic  acid. 
The  fluid  becomes  turbid.  On  the  further  addition  of 
acid  the  turbidity  disappears.  It  reappears  on  neutrali- 
sation. 

2.  Boil  some  of  the  solution,  it  does  not  coagulate. 

3.  Sulphur    is    liberated   in    the    formation    of    alkali    albumin. 

To  2cm  egg  white  add  3  volumes  NaOH  solution.      Mix 


t.;  ... 


134  PROTEIDS. 

thoroughly,  warm  for  a  few  minutes,  then  heat  to  boiling. 
Add  3  drops  acetate  of  lead  solution,  a  black  pp  occurs 
of  lead  sulphide. 

Acid  albumin.  Is  less  readily  formed  with  strong  acids.  To 
some  egg  white  solution  (5  in  50)  add  slowly  one-half  a  volume  of  acetic 
acid  (B.P. ),  agitate,  then  warm  slowly  to  boiling. 

1.  There    is    no  jjp>    on    boiling. 

2.  Neutralise  some  of  the  solution  (litmus)  with  a  few  drops  of  KOH 

diluted  to  one  quarter  with  water,  there  is  a  pp  of  acid 
albumin.     On  further  adding  KOH  the  cloud  disappears. 

3.  Boil  some  of  the  original  solution  briskly  for  a  minute,  add 

lead   acetate  solution,    there   is   no   black   colour. 

Albumoses. 

The  substances  of  interest  in  this  class  are  physiologically 
derived  from  proteids  by  ferment  action  in  the  alimentary 
canal,    and    will    be    taken    later    with    gastric    and    pancreatic 


digestion. 


Compound   Proteids. 


This   very   important  group   contains  : — 

Haemoglobin.  Proteid  +  Hsematin.     Blood. 

Gluco-proteid.  ,,        +  Carbohydrate     (animal    gum,    Land- 

wehr).     .  Mucin    of    saliva. 

Nuclein.  ,,        +  Nucleic   or   Phosphoric   Acid.     Con- 

stituent of   nuclei. 

Nucleo-proteid.  ,,        +  Nuclein.    Chief  constituents  of  cells. 

Mucin  like  subst.  of  bile. 

These  will  be  referred  to  later,  as  far  as  the  scope  of  the 
work  in  class  allows,  under  the  respective  substances  in  which 
they  occur. 


Tables   for   the   rough   separate   recognition    of  proteids  and 

carbohydrates   in   solutions. 

I. — A   solution   of   proteids   and   carbohydrates. 


Xanthoproteic 


Acetic  acid 
and  K.FeCv, 


Heat 


Egg           Scrum 
Albumin 

Globulin  j 

Acid         Alkali 
Albumin 

1  Proteoses 

Peptone 

+                  + 

+ 

-U 

+ 

+ 

pp.              pp. 

pp. 

pp. 

pp. 

PP* 

Coag.        Coag. 

i    Coag. 

— 

— 

—               — 

*  Disappears  on  heating,  returns  on  cooling. 
||.— Coagulable   proteids.         III.— Non-eoag-ulable   proteids. 


Excess  H,0  ... 
MgS04  to  Satn 

Ether  (in  neut- 
ral reaction) 


Egg  A. 

Ser.  A. 

Glob. 

— 

pp. 

— 

— 

pp. 

pp. 

Neutralisa- 
tion 

Biuret 
HN03       . 
IV. — Carbohydrates. 


Acid 

Alkali 

Pro- 
teose 

Pepton 

pp. 

pp. 

— 

— 

Violet 

Violet 

Pink 

Pink 

— 

~~ 

pp. 

Iodine 


Starch 

Dextrin 

Glycogen 

Dextrose 

Maltose    Cane  Sug. 

Blue 

Red 

Port 

— 

—                — 

of  lead 

—            PP- 

— 

— 

— 

Fehliug's  Sol. 
Barfoed's  Reag* 
H.2S04 

Red* 

Redn 

— 

Red11 

and  boi 

L 

Dextrose 

N.B. — To  apply  IV.  in  a  mixture  containing  proteids  acidulate  with  acetic 
acid,  boil  and  filter.  It  is  of  no  consequence  if  peptones  remain  in  the 
solution.  In  performing  the  Biuret  test  employ  one  or  two  drops  of  CuS04 
onlv  to  3C1U  of  fluid. 


CHAPTER  XXL 

SOME  FOOD  SUBSTANCES. 

Milk.  Contains  water  87,  solids  13,  consisting  of  casein 
(ogen)  3,  albumin  0-5,  fat  3-6,  sugar  5,  salts  07.  S.G.  1028 
to   1034  which  is   raised   by  dilution  with  water. 

1.  Reaction — amphoteric. — Fresh  milk  reddens  blue  litmus  and 

turns  red  litmus  blue.  Due  to  acid  and  alkaline  phos- 
phates.     Test  with  litmus  paper. 

2.  Boil    25 cc    milk    in  a   beaker.      It   does    not    coagulate.     A 

scum  forms  upon  the  surface  which  returns  as  t)ften  as 
it  is  removed.  Due  chiefly  to  caseinogen  entangled  in 
proteid  drying  on   exposure  to  the  air. 

3.  Add   a  few  drops   of   dilute   acetic  acid   to   some   milk   in    a 

tube,  a  floccular  pp  of  caseinogen  and  entangled  fat 
results. 

4.  Rennet    (Extract    of    calf's    stomach).     Add   a  few  drops   to 

two-thirds  of  a  tubeful  of  milk,  mix,  digest  at  40°  C.  in 
the  water-bath.  It  will  curdle  in  five  minutes.  From 
this  clot  Whey  exudes  on  standing. 

5.  To  two-thirds  of  a  tubeful  of  fresh  milk  add  3  or  4  drops  of 

a  saturated  solution  of  ammonium  oxalate ;  mix  ;  add  5 
drops  of  extract  of  rennet,  digest  at  40°  C  for  at  least 
half  an  hour.  There  will  be  no  coagulum.  Then  add  a 
few  drops  of  a  2  pc-  solution  of  calcium  chloride.  The 
milk  will  rapidly  coagulate. 


ACTION    OF    REAGENTS.  137 

6.  Prove    the    fermentative   nature   of    curdling   by   mixing  the 

rennet  with  a  little  water  and  boiling  it  before  adding 
it  to  the  milk.  The  milk  will  not  curdle  because  the 
ferment  has  been  destroyed. 

7.  Proteids.     Strained   whey   is    provided    for   you.      Acidulate 

some  with  acetic  acid — no  caseinogen — boil  thoroughly 
there  will  be  very  little  coagulum — lactalbumin — filter 
and    test    the    filtrate    for 

8.  Sugar  in  whey  by  means  of   Fehling  solution. 

9.  Add   ammonium  oxalate  solution   to   some  whey,  a   light  pp 

indicates  calcium  salts.  The  chief  salts.  Test  also  for  P20& 
pg.  160. 

10.  Guaiacum    test.     To    some   fresh  milk    add    a   few    drops    of 

fresh  tincture  of  guaiacum,  agitate,  add  half  a  volume  of 
peroxide  of  hydrogen,  a  blue  colour  due  to  oxygen 
liberated    by   proteids    turning    the    resin    blue. 

11.  Repeat  10  with  boiled   milk — the  blue   colour  is  not  given 

— due    to    changes    in    the   proteid. 

12.  Fat  in  milk.     The  milk  globules  have  already  been  examined 

(Histology).  To  2em  milk  add  2  or  3  vols,  of  ether,  cork 
the  tube,  wrap  it  in  a  damp  cloth  or  folds  of  blotting- 
paper  to  prevent  heating  by  the  hand,  and  shake  thoroughly 
for  30  seconds — there  will  be  no  change.  Add  1  or  2 
drops  of  NaOH  sol.,  shake  again ;  the  ether  will  now 
dissolve  the  fat  and  the  milk  will  lose  its  opacity.  Hand 
the  tube  to  the  laboratory  attendant. 

Estimation  of  cream.  Whole  milk  is  centrifuged  for 
5  minutes  in  a  Watson-Laidlaw  cream  tester.  The  per- 
centage is  read  off  directly  as  solid  cream  in  the  graduated 
tubes  of  the  instrument.  There  should  be  about  12  per 
cent,  present. 


138  SOME    FOOD    SUBSTANCES. 

Vogel's  Lactoscope.  Add  milk  from  a  burette  by  small 
quantities  to  1 00  cc  of  water  in  a  200  cc  flask  until  a  sample 
1  cm  thick  of  the  whole  mixture  held  a  short  distance  from 
the  eye  in  a  glass  test  cell  just  prevents  you  from  seeing 
the  outline  of  a  candle  flame  placed  3  feet  off.  The 
percentage  of  cream  is  ascertained  from  the  number  of 
cc  of  milk  used  by  consulting  Vogel's  table  (Sanderson : 
Handbook  Physiol.  Laboratory,  1873,  pg.  531). 

Flesh.  Muscle.  Take  one-quarter  beakerful  of  lean  minced 
beef  and  half  fill  the  beaker  with  10 pc-  solution  NaCl. 
Extract  in  the  water  bath  at  40°  C.  for  20  minutes  with 
constant   stirring.     Strain   through   muslin. 

1.  Test    the   nitrate   for   proteids. 

2.  Test  the  reaction,  is  lactic  acid  present  %  Uffelmann, 
pg.  142. 

3.  Saturate  some  with  MgS04,  filter  off  the  globulin  and 
test    the   nitrate   for   albumin. 

4.  Free  some  of  the  extract  from  proteids  by  boiling  and 
filtering,  and  test  for  phosphates,  the  most  important  salt, 
by  adding  half  a  volume  of  HN03  and  a  few  drops  of 
molybdate   of   ammonia — heat — a   yellow  pp — P205. 

Coagulation  of  Myosinogen.  (Halliburton  :  Essentials  of 
Chem.  Physiology,  1896.)  An  extract,  which  is  provided  for 
you,  is  obtained  as  follows  : — The  blood-vessels  of  a  rabbit 
which  has  just  been  killed  are  washed  out  with  normal  saline 
through  the  aorta.  The  muscles  are  quickly  removed,  chopped 
up  small  and  extracted  with  5pc-  solution  MgS04  for  24 
hours    in    a    cool    place. 

5.  Dilute  some  of  the  extract  with  4  volumes  of  water, 
and  keep  at  40°  C.  in  the  water-bath.  A  clot  of  myosin 
will    form. 


WHEAT    FLOUR.  139 

6.  To  some  of  the  extract  add  a  few  drops  of  2 pc-  acetic 
acid  (acetic  acid  B.P.  1,  water  16)  a  stringy  2JP  of  myosin- 
ogen  results. 

"Wheat    Flour. 

1.  Make   a    thick    paste   of    wheat   flour,    place    it    in    a    piece 

of  muslin,  knead  in  running  water  until  all  the  starch 
is  removed.  Collect  some  of  the  washings  in  a  beaker, 
and    test    for    sugar    and    starch. 

2.  Examine    the   clot    left   on    the    muslin,   an    adhesive    mass 

of  gluten  (Diabetic  bread),  test  by  the  Xanthoproteic 
reaction. 

Bread.     Contains  approximately   proteids   7,  carbohydrates  55, 
fats    1,    salts    2pa. 

1.  Mascerate  scrapings  of  crust  in  water  and  test  the  solution 

for  sugar,   starch,  and  dextrin. 

2.  Do    the    same    with    the    crumb. 


CHAPTER   XXII. 
DIGESTION. 

Saliva.     See  starch.      Collect    saliva    as    already    directed, 
pg.   124. 

1.  To    saliva    add    2     volumes    water,    then   a    few   drops    of 

acetic  acid.     A   white   stringy  pp   of  mucin  falls. 

2.  To    saliva   add    a  drop    of   ferric  chloride  solution.     A  red 

colour  results,  which  is  discharged  by  HgCl2 — Potassium 
sulpho-cyanide. 

Gastric  Digestion. 

Arrange  the  following  digestions  at  40°  C.  in  the  water-bath ; 
in  tubes  :    examine   30   minutes   afterwards  : — 

1.  Water  +  a   small    flocculus   of  Fibrin. — No  change. 

2.  A  0-2 pc-  solution   HC1.  +  Fibrin. — The  fibrin  swells. 

3.  Water  +  a  little  Pepsin   (Extract)1  +  Fibrin. — No    effect. 

4.  Dilute  IICl  +  Pepsin  +  Fibrin. — The  fibrin  is  dissolved. 

Whilst  waiting,  proceed  with  the  following  : — 

Products  of  Gastric  Digestion. 

Fill  a  beaker  one-third  full  of  the  fibrin  which  is  provided 
for  you  and  which  has  been  swollen  in  0*2 pc-  HCl,  to  it  add 
another  volume  of  dilute  acid  and  raise  the  mixture  and 
maintain  it  at  40°  C,  then  stir  in  5 cc  peptic  extract  (Benger's). 
In  a  few  minutes  the  gelatinous  mass  will  become  fluid. 
Strain  through  filter  paper  to  remove  coarse  impurities,  and 
return  it  to  the  beaker  and    continue   the  digestion. 

]  Neutralised  Liquour  pepticus  (Benger)  or  a  glycerin  extract. 


GASTRIC    DIGESTION.  141 

1.  Take  a  sample  and  boil  it. — There  should  be  no  coagulable 

proteid. 

2.  Neutralise    another    portion    carefully    using    litmus    as    an 

indicator. — There  will  be  a  pp  of  acid  proteid. 

Add  some  warmed  dilute  IIC1  to  the  digestion  to  fill  the 
beaker,  and  test  samples,  as  follows,  from   time  to   time. 

As  soon  as  test  (2)  gives  markedly  diminished  results  and 
the  tests  (3)  and  (4)  are  well  marked,  set  two-thirds  of  a  beaker- 
ful   aside,  neutralise  and   label   it   (A). 

To  the  remainder  add  one  volume  warm  dilute  HC1,  and 
continue   the  digestion. 

3.  HiSTOg  a  few  drops. — A  white  pp  which  disappears  on  heating 

and   returns  on   cooling  (proto-proteose). 

4.  Two    drops  of    acetic  acid    and    a    few  of    ferrocyanide  of 

potassium  solution. — A  white  pp  which  disappears  with 
heat  and  returns  on  cooling  (proto-  and  deutero-proteose). 

5.  Add  one-quarter  volume    ISTaOH   and    one  or    two  drops   of 

CuS04  solution. — Biuret  reaction.    Pink  indicates  proteoses 
as  well  as  pepton. 
When  test  5   is    the  only  reaction   given,  then    nothing   but 
pepton   is  present.     This    stage   will,  however,  not   be   reached 
during  the  time  which  is  at  the  disposal  of  the  class. 

6.  Saturate  some  of  the  fluid  with  Am2S04  whilst  boiling  and 

first  acidify  by  means  of  a  little  acetic  acid,  then 
neutralise  with  NaOH  solution,  boiling  after  each  addition  ; 
filter  and  test  the  filtrate  for  pepton,  by  adding  four 
volumes  of  NaOH  solution  and  then  a  few  drops  of  CuS04. 
Boiling  in  different  reactions  has  the  effect  of  precipi- 
tating the  proteoses  completely.  The  excess  of  alkali 
in  the  Biuret  test  is  to  set  aside  the  effect  of  the 
Am2S04  which  would  interfere  with  the  reaction. 


142  DIGESTION. 

Proteoses.  The  following  reactions,  which  may  be  regarded 
as  the  best  marked  class  distinctions,  are  all  that  can  be 
attempted  here.  Fluid  (A)  which  has  stood  after  neutralisa- 
tion should  be  filtered  through  double  paper,  the  filtrate 
will  be  free  of  acid  proteid  and  nearly  clear.  It  contains 
proteoses   and    pepton1. 

Primary   proteoses    (pro to-  and    hetero-proteose) 

1.  Saturate  a  tubeful  with  NaCl  crystals,  a  pp  separates, 
consisting  principally  of  primary  proteoses — much  of  this  pp 
is    carried   to   the   top   with   the   froth — filter. 

Secondary   proteoses    (deutero-proteose). 

2.  To  the  filtrate  from  (1)  which  is  quite  clear,  add  a  few 
drops  of  acetic  acid,  a  further  pp  will  form,  consisting 
mainly  of  deutero-proteose,  but  containing  a  little  proto- 
proteose   as    well. 

Note. —  The  primary  proteoses  are  formed  first  and  the 
secondary  proteoses  next.  The  relationship  which  these  sub- 
stances are  supposed  to  bear  to  the  parallel  chains  of  cleavage 
products  of  the  proteid  molecule,  known  as  the  hemi-  and 
anti-groups,  may  in  brief  be  expressed  as  follows : — Proto- 
proteose  is  stated  to  be  the  first  link  in  the  hemi-  and 
hetero-proteose  the  corresponding  link  in  the  anti-chain, 
whilst   the   next   link    in   each    is    a   deutero-proteose. 

9  Recognition  of  free  Hydrochloric  and  Lactic  acids. 
Perform  the  following  reactions  with  watery  solutions  of  the  acids, 
by  adding  them  drop  by  drop  to  -]-  tubeful  of  the  reagent: — 

HC1  0-1  *e-  C„H6O30-0111c- 

1.  Congo  red  (Hosslin)      ...       Blue 

2.  Carbol-iron  (UfFelmann)  Pale  yellow. 
Carbol-iron  test. — To  10  cc  of  a  4pc>  solution  of    carbolic  acid 

add  20 cc  distilled  water  and   1  Liquor  Ferri  perchloridi  B.P. 

l  What  is  the  definition  of  a  pepton? 


PANCREATIC    DIGESTION.  143 

Pancreatic    Digestion. 

The  Amylolytic  ferment  action  has  already  been  studied 
under  starch. 

Proteolytic   action. 

Make  an  artificial  digestive  fluid  by  adding  2CC  Liquour 
pancreaticus  (Benger)  to  100 cc  of  a  lpc-  solution  bi-carbonate 
of  soda  at  40°  C.  in  a  beaker.  Add  a  few  shreds  of  fibrin 
and  observe  that  these  gradually  diminish  in  bulk  without 
swelling  (erosion).  The  whole  process  is  much  hastened  by 
previously  soaking  the  fibrin  in  the  bi-carbonate  of  soda, 
but    is    slower    than    artificial    gastric    digestion. 

Perform  the  same  tests  as  in  the  case  of  gastric  digestion, 
and  note  the  absence  of  proto-proteoses.  The  digestion  is  to 
be  pushed  much  further  than  in  the  former  case.  The  fluid 
is  to  be  filtered  from  coarse  impurities  when  all  the  solid 
fibrin  has  disappeared,  and  if  the  changes  languish  diluted 
with  bi-carbonate  of  soda  solution.  Note  the  persistent 
bitter  taste  of  the  solution — (Albumoses).  After  all  traces  of 
albumoses  have  disappeared,  or  before,  withdraw  any  sediment 
which  may  form  with  a  pipette  and  examine  under  the  microscope 
for  leucin  small  yellowish  balls  and  tyrosin  colourless  acicular 
crystals. 

Whilst    the    above    is    taking    place  : — 

Take  half  a  tubeful  of  milk,  add  an  equal  volume  of 
1 pc-  solution  bi-carbonate  of  soda  and  4  drops  of  pancreatic 
extract. — Digest. — Note  the  bitter  taste  that  soon  appears 
and  which  may  be  taken  as  an  indication  of  the  digestive 
change    which    the    proteids    of   the    milk    are    undergoing. 

Steaptic   action — fat  splitting  ferment.     The  demonstration 
of    this    action    does    not    lend    itself    readily    to    class    work. 


CHAPTER  XXIII. 
BLOOD. 

Composition  of  Blood  (Approximate  average,  human). 
Water  77 p%  solids  23**.  Of  the  latter  haemoglobin  14 
proteids    7*5,    salts    1*5    (urea    0-02,   glucose    (H2). 

Colour.    Arterial — scarlet ;  Venous — purple. 

Reaction.  Alkaline.  Due  to  Na2HP04  and  sodium  car- 
bonate. Place  a  drop  on  red  litmus  paper  moistened  with 
normal  saline,  and  after  15  seconds  wash  with  the  same. 
Observe  the  colour  of  the  stain. 

Specific  gravity.  1035-1068.  Estimate  it  by  Roy's  method 
(Lloyd-Jones).     The    following    are    required  : — 

(a)  A    stock    of     standard    solutions     (SS)     of    glycerin     and 

water   of    specific   gravities   varying    from    1030  to   1080 
by  steps  of   5   degrees. 

(b)  Glass     tubes     drawn     to     capillary    ends,     of     which     the 

terminal    5 mm    are    bent    at    right    angles. 

(e)    A    3    drachm   phial   or   an    8 cc  tube. 

Process  : — Commencing  with  88  of  a  S.G.  1045  in  the  phial, 
fill  the  capillary  tube  for  2cm  with  blood  from  your 
finger.  Do  not  squeeze  the  finger  in  obtaining  it. 
Plunge  the  capillary  point  into  the  fluid  in  the  phial, 
holding  the  bent  part  horizontally,  and  expel  a  small 
drop.      If    the    drop    falls    or    rises,    the    S.G.    of    the    88 


PROTEIDS    OF    SERUM.  145 

is  less  or  greater  than  that  of  the  blood.  Change 
the  SS  accordingly,  until  the  drop  neither  rises  nor 
falls    during    the    first    moments    after    ejection. 

The  S.G.  of  blood  varies.  It  is  highest  in  the  male  sex 
and  at  birth  ;  is  lowest  from  the  ages  of  35  to  45  years. 
Falls    with    hunger,  pregnancy,  food    and    drink. 

Proteids    of  Serum. 

Blood  serum.  Contains  approximately  7 pe-  proteids  consisting  of 
4  parts  serum-albumin  and  of  3  parts  serum-globulin  (para-globulin). 

Sheep  or  ox  blood  is  allowed  to  clot  in  the  vessel  into  which  it 
lias  been  run  from  the  animal  in  the  slaughter  house  ;  on  standing  the 
clot  contracts  and  squeezes  out  the  serum.  If  any  water  be  present 
in  the  vessel  at  the  time  the  blood  is  run  in  the  serum  will  be  tinged 
with  the  colouring  matter  of  the  corpuscles. 

Dilute  10 cc  serum  to  70cc  (=  1  pc-  proteid  approximately) 
with  water  and  repeat  the  reactions  performed  on  egg 
albumin. 

Difference   between    egg   and  serum   albumin. 

Serum  albumin  is  more  soluble  in  nitric  acid.  Egg 
is  coagulated  by  ether  in  a  neutral  reaction,  whilst 
serum    is    not. 

Relative  sensitiveness  of  some  of  the  proteid  reactions. 

Dilute  some  of  the  above  reputed  1  pc-  solution  to 
50  cc  with  normal  saline  =  0-1  pc-  solution  (a);  repeat  this 
operation  successively  with  5  cc  of  (a)  =  0*01  pc-  (b)  ;  5  cc 
of  (b)  =  0-001  p-°-  (c);  5ccof  (c)  =  0-0001  pc-  (d)  ;  5  cc  of 
(d)  =  0-00001  pc-  (e). 

Take  5  tubes,  number  them  from  1  to  5,  place  in  each 
of  them  2  cm  of  HN03.  Do  this  by  means  of  a  pipette 
so  as  not  to  wet  the  sides  of  the  tubes.  Next,  with  the 
pipette  pour  two  volumes  of  proteid  solution  (e)  upon 
the  acid  in  tube  No.  5,  being  careful  to  avoid  mechanical 


146  BLOOD. 

mixture    of    the    two    fluids.      In   a  similar    manner  add 

two  volumes  of   (d)   to  tube  No.  4,  and  so  on  with  the 

remainder.      Empty,    rinse    and    blot    off  fluid   from  the 
pipette  between  each  addition  of  proteid  solution. 

Note  carefully  the  lapse  of  time  by  your  watch  between 
the  moment  of  bringing  the  two  fluids  together  and  the 
appearance  of  a  cloud  at  their  plane  of  junction. 

Test  the  solutions  (c),  (d),  and  (e)  with  tests  5  and  6  for 
proteids  in  the  same  way,  being  careful  to  use  the  same 
quantity  of  reagent  in  each  case,  and  determine  their  relative 
sensitiveness.     Construct  a  table  of  the  results. 

Demonstrate  the  presence  of  a  globulin  in  serum. 

To  some  serum  which  has  been  diluted  with  2  volumes 
of  water  add  an  equal  quantity  of  saturated  solution  of 
Am2S04,  the  globulins  will  be  ppd.  Filter  and  care- 
fully scrape  the  residue  off  the  filter  with  a  knife,  and 
mix  it  with  some  water,  the  pp  will  probably  be 
re-dissolved,  enough  neutral  salt  being  present  to  do  so. 
If  not,  add  a  little   10 pc-  NaCl  solution  until  it  does. 

Test  the  filtrate  for  serum  albumin  (a)  by  boiling,  and  (6) 
by  saturating  with   Am2S04. 

Proteids  in  blood,  plasma.     Coagulation  experiments. 

Besides  those  already  recognised  in  the  serum,  plasma 
contains  fibrinogen. 

(A)  Salted  plasma. 

Obtained  by  mixing  blood  as  it  flows  from  the  blood-vessels 
of  an  ox  or  sheep  with  half  its  volume  of  a  saturated  solution 
of  XaS04,  and  then  centrifuging. 


PLASMA.— SUGAR.  147 

1.  Dilute  some    with    10   volumes   water   in  a  tube   and    place 

it  in  the  water-bath  at  37°  C.  to  hasten  coagulation. 
In  about  twenty  minutes  the  fluid  will  turn  into  a  perfect 
but  thin  jelly.  Note  the  quivering  of  the  jelly  when 
the  vessel  is  slightly  shaken.  Let  the  clot  rest  until 
the  next  day,  when  it  will  be  found  to  have  shrunk 
considerably. 

2.  Heat    some    undiluted    neutralised     plasma    slowly    in    the 

water-bath,  and  determine  the  temperature  at  which 
coagulation  first  appears.  Fibrinogen  coagulates  at  about 
56°  C. 

(B)   Oxalated  plasma.      {Decalcified  plasma.) 

To  blood  as  it  flows  from  the  blood-vessels  of  an  animal 
one  quarter  of  its  volume  of  a  1  pc-  solution  of  potassium 
oxalate  in  normal  saline  is  added  (Arthus  and  Pages).  The 
blood  is   then   centrifuged. 

To  8  cm  of  this  plasma  add  5  drops  of  a  2  pc-  solution  of 
calcium  chloride.  Place  in  the  water-bath  at  37°  C.  Coagula- 
tion will  take  place. 

Sugar  in  the  blood.  Boil  some  fresh  serum  which  has  been 
diluted  with  2  volumes  of  water,  and  slightly  acidulated 
with  dilute  acetic  acid.  Coagulate  the  proteids  com- 
pletely, which  will  take  five  minutes ;  filter  off  as  much, 
perfectly  clear  fluid  as  you  can,  and  test  a  quarter 
tubeful  of  the  filtrate  by  boiling  it  with  about  8  drops  of 
Fehling.  If  the  reduction  is  not  very  well  marked  let 
the  tube  stand,  and  at  the  end  of  twenty  minutes  a 
distinct  pp  of  sub-oxide  will  collect  at  the  bottom,  showing 
the   presence   of  a  reducing   sugar. 


148 


BLOOD. 


12  Enumeration  of  the  Red  Blood  Corpuscles. 

Thoma-Zeiss  Hsemaeytometer.— This  consists  of  : — 

1.  Dilution  pipette,  or  mixer,  having  a  bulb  containing  a  glass 

bead  for  mixing.  The  stem  is  graduated  from  0*1  to  1, 
and  to  101  above  the  bulb. 

2.  Counting  chamber,  a  cell  with  an  outer  rim,  and    a  central 

platform,  the  latter  ruled  in  squares ;  each  square  has  a 
side  of  1/20 mm,  and  hence  an  area  of  1/400 mm  square. 
The  film  of  fluid  between  the  glasses  is  0-1 mm  thick 
when  the  chamber  is  covered,  and  consequently  the 
portion  over  each  square  is  1/4000  of  a  cubic  millemetre. 
Groups  of    16  squares  are  separated   by  additional    lines. 


Fig.  15.  Thoma-Zeiss  Hemacytometer,  a  and  b  the  counting  chamber,  in  which 
W  is  the  outer  rim  upon  which  the  cover  D  rests.  B  central  platform,  and  r  the  circular 
trench  which  separates  it  from  D.  c  divisions  on  B  (x  30).  S  M  mixing  pipette  for  red 
blood  cells. 

Process  : — 

1.  Puncture  the  finger  freely. 

2.  Draw   blood  up  in   the  mixer  to   0*5   or   1   on   the  stem   of 

the  pipette.     Wipe  the  point  dry. 

3.  Draw  in  3  pc-  salt  solution  to  the  mark   101   above  the  bulb, 

and    mix   the   fluids   by  shaking. 


HEMACYTOMETER.  149 

4.  Expel  the  fluid  that  fills  the  stem  and   wipe  the  point. 

5.  Blow  a  small  drop  of    the  diluted    blood   on  to    the  central 

platform   of  the  counting   chamber,  and   apply  the  cover. 
On  pressing  the  latter  down 

6.  Newton's  rings  should    be  seen  between  the  rim  of  the  cell 

and  the  cover. 

7.  Let    the    preparation  rest    for    two    minutes,   the    blood  cells 

settle  on  the  floor  of  the  chamber. 

8.  Count   the  cells    in    at    least   16  squares.     Cells   astride  the 

lines  are  to  be  counted   on  two  sides  of  each  square  only. 

Calculation  : — 

Number  of  cells  (200  V)  x  4000  x  100      R.B.C.  in  1   cubic  milli- 
Number  of  squares  (16  1)  metre  of  blood. 

Oliver's    Haemaeytometer   (used  in  the  darkened  room). 

(1)  A  graduated  flattened  tube,  (2)  an  automatic  blood  measurer  to 
which  (3)  a  mixing  pipette  is  adjustable  for  washing  it  out,  (4)  a  candle 
(Christmas  candle),  (5)  a  bottle  of   Hayem's  solution  (see  pg.  61). 

Process :— ( 1 )  Dry  the  measurer  by  drawing  darning  thread  through  it 
with  a  needle.  (2)  Prick  your  finger  and  fill  the  measurer  by 
touching  the  drop  of  blood.  (3)  Attach  the  mixer,  rilled  with 
Hayem's  fluid,  to  the  measurer  by  means  of  the  rubber  tube  and 
wash  the  blood  thoroughly  into  the  graduated  tube.  (4)  Mix 
thoroughly  by  inverting  three  times.  In  withdrawing  the  thumb 
draw  it  against  the  edge  to  avoid  loss  of  fluid.  (5)  Place  the 
lighted  candle  ten  feet  off.  (6)  Grasp  the  tube  by  the  flat  sides 
framing  it  between  the  thumb  and  fore-finger,  hold  it  close  to  the 
eye,  and  look  through  its  long  diameter  at  the  flame.  (7)  Add 
small  quantities  of  Hayem's  fluid,  repeating  (4)  until  the  eye  can 
distinguish  the  first  appearance  of  a  transverse  line  of  light.  (8)  Read 
the  graduation  touched  by  the  upper  edge  of  the  column  of  mixture 
and  calculate  as  follows  : — 100  degrees  correspond  to  the  assumed 
normal  of  5,000,000  cells  per  cubic  millimetre.  Each  degree  above 
or  below   this    indicates  50,000  cells  above  or    below    the  normal. 

The  W.B.C.  in  a  state  of  health  do  not  affect  the  readings. 

This  instrument  has  been  standardised  by  means  of  the  Thoma- 
Zeiss  instrument. 


150  BLOOD. 

Enumeration   of  White   Blood   Cells. 

Use   the  Thoma-Zeiss   instrument   with   a   sj)ecial  pipette. 

Dilute  the  blood  to  10  or  5  pc-  in  the  same  manner  as  for 
the  red  cells  but  with  0*3  pc-  solution  glacial  acetic  acid,  and 
proceed  as  before.  1  pc-  dilution  will  answer  if  the  white 
cells  are  very  numerous,  provided  many  fields  of  16  squares 
are  counted.  0'6  pc-  jSTaCl  solution  coloured  with  gentian  violet 
may  then  be  used    as  a  diluent. 

Enumeration  of  Blood  Platelets.  Use  the  same  instru- 
ment and  dilute  the  blood  with  Bizzozzero's  fluid  (methyl  violet 
1   in  5,000  of  normal   saline)   to   0-5  pc- 

The  operation  must  be  carried  out  quickly,  as  the  platelets 
tend  to  adhere  to  the  glass  surfaces  and  to  each  other.  They 
must  be  carefully  sought  for.     Film  preparations  show  them  best. 

Estimation  of  Hsemoglobin.  (Blood  contains  normally 
from    13   to   14 pc) 

Gowep's  Instrument  is   composed  of  : — 

1.  Standard  colour  tube  of  the  tint  of  a   1  p-c-  solution  of 

normal  blood. 

2.  Graduated   tube  for  diluting  blood. 

3.  Block  for   supporting  these  side  by  side. 

4.  A   20   cubic  millimetre  pipette. 

Process : — 

Place  a  drop  of  distilled   water  in   (2). 

Fill  (4)  up  to  the  mark   with  blood,  wipe  the  point,  and  expel 

the  contents  into   (2). 

Add  distilled  water  to  this  drop  by  drop,  with  agitation,  until 

a  tint  exactly  matching  that  of  the  standard  is  obtained. 

According    to    the    dilution    required    so    is    the    quantity    of 

Hb02.     If  the  desired   tint  is  obtained  when  the  fluid  in   the 


1 1  &JMOGLOBINOM  ETER.  1  5  1 

graduated    tube   stands   at    90,  then    there    is   present   90 p,c-  of 
the  normal  quantity  in  the   blood,  e.g.,  of  lJf.pc- 

Should  the  fluid  reach  the  graduation  120  then  there  is 
more  by  20  pc-  than  the  normal   amount. 

Oliver's   Haemoglobinometep.       (To  be  used  in  a  darkened  room.) 

(1)  Automatic  blood  measurer,  (2)  mixing  pipette,  (3)  the  blood  cell 
and  cover  glass,  the  latter  of  low  grade  blue  glass,  (4)  a  set  of 
standard  colour  grades,  (5)  riders,  (6)  camera  tube,  (7)  light 
(Christmas  candle),  (8)  bottle  of  antiseptic  fluid,  lancet,  needle  and 
thread. 

Process: — (1)  Dry  the  blood  measurer  by  drawing  darning  thread 
through  it  with  a  needle.  (2)  Apply  the  point  to  the  drop  of  blood 
exuding  from  a  pricked  finger  ;  it  will  fill  itself.  There  must  be  no 
break  in  the  column  of  blood.  Dry  the  ends  with  a  finger  tip. 
(3)  Fill  the  mixing  pipette  with  distilled  water,  and  fit  it  on  the 
measurer.  (4)  Expel  the  blood  into  the  blood  cell  by  pressing  water 
through  drop  by  drop.  Stir  with  the  handle  of  the  measurer  and 
use  it  as  a  guide  for  adding  the  last  drops  required  to  fill  the  cell 
exactly  level  with  the  edge.  Do  not  fill  to  a  convexity.  (5)  Apply 
the  cover,  a  small  bubble  should  form,  then  the  cell  has  not  been 
overfilled.  (6)  Place  the  blood  cell  by  the  side  of  the  standard  colour 
grades  under  the  camera  tube,  so  that  they  are  seen  through  separate 
apertures  at  the  bottom  on  looking  down  the  tube.  (7)  Place  the 
lighted  candle  at  about  10 cm  equidistantly  from  the  blood  cell  and 
the  standard.  (8)  Make  the  observation  by  looking  through  the  tube 
for  not  more  than  10  seconds  at  a  time,  and  in  order  to  resensitise 
the  retina,  if  there  is  any  fatigue,  close  the  upper  opening  of  the 
tube  with  your  finger,  and  glance  for  a  few  moments  through  the 
green  glass  at  the  light  before  making  another  observation.  Match 
the  blood  tint  exactly  with  one  of  the  blood  standards.  Physiological 
riders  are  supplied  of  nine  degrees  between  standard  grades.  If  the 
blood  tint  lies  between  two,  superpose  riders  on  the  lighter  tint 
until  a  match  is  obtained.  To  balance  the  glass  of  the  rider  a 
colourless  slip  is  placed  over  the  cover  of  the  blood  cell.  A  descrip- 
tion of  this  instrument  is  given  because  it  rests  upon  (1)  the 
recognition  of  the  irregular  variation  of  the  blood  tint  on  dilution, 
and  (2)  upon  the  employment  of  the  delicate  colour  discrimination 
method  introduced  by  Mr.  Lovibond,  of  the  Tintometer  Company, 
which  admits  of  many  applications.  A  power  of  acutely  discriminating 
differences   of   tint   is   required   in  the   observer. 


152 


BLOOD. 


13  Spectrcscopical  examination  of  the  blood.  Use  a 
small  direct  vision  spectroscope  suitably  supported.  Keeping 
the    red   end     to    the     left    the    spectrum    is    to    be    sharply 

focussed  by  adjusting 
the  draw -tube.  The 
slit  is  to  be  reduced 
by  means  of  the  milled 
head  at  the  other  end 
of  the  instrument,  short 
of  seeing  the  horizontal 
lines  caused  by  the 
irregularities  of  the 
jaws  of  the  slit.  Burn 
biborate  of  soda  in  the 
flame  to  get  the  ISTa 
line. 


Draw  a  chart  of  the 
spectrum  by  marking 
the  position  of  the  sod- 
ium line  and  the  limits 
of  the  colours  by  vertical 
lines,  continuing  them 
on  the  paper  to  serve  as 
guides  in  plotting  below 
each  other  the  follow- 
ing absorption    spectra. 

FlG.  16.   Direct  vision  spectroscope  S  on  a  wooden     Examine        the         blood 
stand.     P  wedge-shaped  bottle  for  blood  solutions.  _ 

W  platinum  wire  with  a  borax  bead  for  the     solutions       in       wedged- 

M,,liumline-  shaped   bottles1. 

Solution   of  blood.     Make   a   2pc-   solution  of  ox  or  sheep's 
blood   in   water.      Or   you  may  add  one  or  two  drops  of  your 


'.Made  for  the  author  by  the  York  Glass  Company. 


HEMOGLOBIN    SPECTRA.  153 

own  blood  to  a  wedgeful  of  water.  Note  in  each  case  the 
influence  of  the  quantity  of  the  pigment  upon  the  absorption 
bands  by  moving  the  wedge  bottle  across  the  slit  of  the 
instrument.     This   has    the   same   effect   as    dilution. 

SPECTRA    OF    HAEMOGLOBIN    COMBINED    WITH     GASES    OR    ALONE. 

1.  Oxyhemoglobin  —  Hb0.2.      Determine  the  relationship  of  the 

two  bands  between  D  and  E  to  the  Na  line  and  to  the 
colours.  These  are  respectively  known  as  the  a  and  /3 
bands.  Compare  with  (3)  and  (8). 
Note  : — Examine  the  line  of  junction  of  the  closed  fingers 
before  a  strong  light,  the  single  band  of  a  thick  solution 
of  Hb0.2  will  be  seen. 

2.  Reduced    op    Gas-free    Haemoglobin— lib.         Add       a      few 

drops  of  (NH4)2S  or  of  Stokes'  fluid.1  The  bands  of 
Hb0.2  will  gradually  fuse  into  each  other.  This  single 
band  is  less  dark  than  the  other  two.  Try  and  restore 
HbO.,  by   shaking  with   air. 

3.  Capbon-monoxide   Haemoglobin— HbCO.     Coal  gas  (4pc   CO) 

is  passed  for  20  minutes  through  blood  solution.  Note 
its  cherry-red  colour.  The  two  bands  closely  resemble 
those  of  HbOo.  Observe  the  relationship  of  the  D 
band  to  that  line.  Add  (NH4),S  there  is  no  result. 
This   compound   resists   putrefaction   for  a    long  time. 

4.  Neutpal     Met-Haemoglobin— Met-Hb.      Add    2    or   3   drops   of 

a  10 pc-  solution  of  ferricyanide  of  potassium  to  a 
bottleful  of  solution  Hb02 — mix.  The  colour  turns  of 
a  brownish  tint.  A  characteristic  band  appears  in  the 
red  and  another  dim  band  can  just  be  recognised  to 
the    green    side    of    E>. 

i  Ferrous  sulphate  2g,   tartaric   acid  3s,   mix  and   preserve  dry   for  use.     When 
required  add  100  ec  water,  and  add  ammonia   to  slight   alkalinity. 


154  BLOOD. 

5.  Alkaline    Met-Haemoglobin.— To    (4)    add    2    or    3    drops     of 

strong    ammonia.     The   colour    turns    ruby   red.     A  band 

on   the   red   side    of   and  cut  by  D   and   another  in  the 

green. 

A    blood-stained    rag   is   given    to  you.     Examine   by  cutting 

out    a    small    piece    of    the   stain   0*5 sqcm  and  steeping  it  in  the 

least    quantity    of    normal    saline    on    a    slide.     Search    for    (1) 

red  blood  cells,  (2)   absorption  bands  (microspectroscope  if  the 

quantity    is    very    small),    (3)    haemin    crystals. 

DECOMPOSITION    SPECTRA    OF    HAEMOGLOBIN. 

6.  Acid  Haematin.     To   a   bottleful   of  blood   solution  add  ten 

drops  of  acetic  acid.  Slight  warming  hastens  the  change. 
The  heat  of  the  name  near  the  bottle  on  the  spectro- 
scope stand  will  do  this.  Note  the  alteration  in  the 
colour — brown  tint.  One  band  well  in  the  red  is 
characteristic    with    some   obscuration    of    the   green. 

7.  Alkaline  Haematin.     To   a   bottleful  of  blood   solution   add 

ten  drops  of  a  10pc-  solution  NaOH.  Warm  in  a  tube 
to  hasten  the  change.  One  broad  band  to  the  red  side 
of  and  cut  by  the  D  line.  Haematin  in  ethereal  solution 
gives   a   four-band   spectrum. 

8.  Reduced  Haematin.     (Stokes'  reduced  haematin,  haemo-chro- 

mogen.)  Treat  (6)  with  a  few  drops  of  (NH4)2S.  It  changes 
to  reduced  haematin.  Two  bands  in  the  green — the  band 
nearer  T)  is  the  darker  of  the  two  and  persists  longest 
with   dilution. 

9.  Haematoporphyrin    in    acid    solution.      Add    four    drops    of 

undiluted  defibrinated  blood  to  3 cm  strong  H2S04  in  a  tube, 
agitate.  The  solution  must  remain  clear,  and  present  a 
deep  cherry-red  colour  (iron-free  haematin).  Two  bands — 
a  thin  one  to  the  red  side  of  and  touching  D,  and  a 
broader  one  on  the  other  side  of  and  shading  off  towards 
D.     The   latter   persists   longest   with   dilution. 


CHAPTER    XXIV.  14 

BILE. 

Liver    bile    2 1>c- ,    bladder  bile    1 2 1,c-   solids.      The   difference    is 
due    to    concentration    in    the    gall    bladder    and    ducts, 
where   also   mucinous   substances   are   added. 
Use  bile  from  the  gall  bladder  of  the  ox,  sheep,  or  pig. 

1.  Note   its  ropiness   or   viscidity.     Due   to  mucin  and  neucleo- 

albumin.  Add  a  few  drops  of  dilute  acetic  acid  a 
stringy  pp  falls.  (A  mixture  of  mucin  and  neucleo- 
albumin.) 

2.  Proteids.       Dilute    bile   with    3    volumes    of    water,    boil — no 

result. 

3.  Bile    salts.       Glyco-cholate    and    tauro-cholate    of    soda    and 

salts   of  fellic   acid. 

•a.  Pettenkofer's  reaction.  To  some  bile  add  two-thirds  volume 
of  H2S04  so  slowly  that  the  temperature  does  not  rise 
above  60°  C,  then  add  3  to  5  drops  of  cane  sugar 
syrup  (1  in  5),  agitate,  a  red  colour  passing  into  violet 
results.  The  acid  simultaneously  produces  furfur ol  from 
the  cane  sugar  and  liberates  the  cholic  acid,  which,  reacting 
upon  each  other,  yield  the  colour.  The  violet  tint  must  be 
present. 
This  reaction  is  not  specific  in  the  urine  as  other  substances 

give    it    also.       (Udranszky   mentions    amongst    others  proteids, 

cholesterin,    phenol,    turpentine,    salicylic    acid,   pyrogallol,    and 

morphin.) 

b.  Strasburger's  modification  of  the  above  test.  Mix  the  bile- 
containing  fluid  with  syrup,  dip  pieces  of  blotting  paper 
into    it.       Dry,   then    touch    with    H2S04.       After    fifteen 


156  BILE. 

seconds  the  stain  appears  violet  by  transmitted  light. 
The  paper  may  be  placed  between  two  glass  slips  and 
examined  spectroscopically.  Two  faint  bands  to  the 
blue  side  of  D ;    one  close  to  D,  the  other  near  the  blue. 

c.  Surface  tension  test  (Hay).  Sprinkle  a  little  flowers  of 
sulphur  on  the  surface  of  the  fluid,  it  will  sink.  Make 
a  control  test  with  water  and  compare  its  behaviour 
with   water  to  which  a  little  bile  has  been  added. 

4.  Pigments.     Bili-rubin   and   bili-verdin  are   changed  by  oxida- 

tion. 

Gmelirfs  reaction.  On  mixing  the  fluid  with  HNO.,  con- 
taining  nitrous  acid — let  two  pools  of  the  fluids  flow 
together  on  a  plate — a  play  of  colours  occurs  passing, 
through  green,   blue,   violet,    red   to  fawn. 

If  the  reaction  be  performed  in  a  tube  the  colours 
will  be  produced   in   successive  layers  above  each  other. 

5.  Cholesterin: — Obtained   from   gall   stones.       Dissolve   a   small 

pinch  of  powdered  gall  stone  in  3cm  of  equal  parts 
ether  and  alcohol,  pass  through  a  dry  filter.  Place  2, 
drops  on  a  glass  slide,  cover,  and  let  it  evaporate  slowly. 
Irregular  cholesterin  crystals  will  separate  out.  When 
regularly  formed  the  crystals  are  rhombic  plates  with  a 
corner  broken  out. 

When  dry  flow  under  the  cover  a  mixture  of  H2S04 
with  a  quarter  volume  of  water,  then  a  small  quantity 
of  iodide  of  potassium  solution.  (Salkowski's  Practical  in.) 
The  cholesterin  crystals  colour  brown,  violet,  or  even 
blue,   and   are   partially   dissolved. 


CHAPTER    XXV.  15 

HEALTHY   URINE. 

Contains    roughly  4 pc-   of   solids,    2    parts  of  which  are  urae 
and    1    part    NaCl. 

Quantity   in    24    hours   about    1,500 cc   or    52|    ounces. 

Colour.  Pale  straw  to  a  deeper  tint.  Three  pigments  are 
usually  recognised.  (1)  Urobilin,  (2)  Indican  =  indoxyl 
sulphate  of  potassium,  and  (3)  Uroerythrin.  The  first 
may  be  regarded  as  the  normal  pigment  derived  from 
the  blood.  The  second  a  form  of  indigo  derived  from 
the  alimentary  canal,  and  the  third  as  only  occasionally 
present  in  recognisable  quantity.  The  latter  tinges  the 
pp  of  urates  in  feverish  conditions  of  a  rose  or  brick 
red  colour.  A  chromogen  of  unknown  nature  is  also 
said   to   be   present. 

Test  for  Indican.  To  7cm  urine  add  5  drops  of  HC1,  mix, 
then  add  nearly  an  equal  volume  of  HN03.  The  colour 
will  darken  above  the  last  acid  to  a  red-brown — 
indigo  red  1    A  tendency  to   violet   indicates  indigo  blue. 

Odour. —Urinous. 

Reaction. — Usually  faintly  acid.  Due  mainly  to  acid  sodium 
phosphate.  May  become  alkaline  during  the  period  of 
digestion,  due  to  alkaline  sodium  phosphate  from  food. 
This  alkalinity  is  transient.  The  acidity  may  be  quanti- 
tatively stated  in  terms  of  a  normal  sodium  carbonate 
solution     if     thought    desirable.     After    urine    has    been 


158  HEALTHY    URINE. 

passed,  it  will  change  in  reaction.  According  to  tempera- 
ture and  circumstances,  often  in  24  hours  it  undergoes, 
the  alkaline  fermentation  by  the  micrococcus  urea,  which 
breaks  up  urea,  yielding  carbonate  of  ammonia  in  the 
solution.  This  change  may  occur  in  the  bladder  when  a 
dirty  catheter  is  employed  to  draw  off  urine.  Such  urine 
becomes  turbid,  smells  of  ammonia,  a  surface  scum  forms, 
and  a  heavy  white  pp  of  phosphates  of  Ca  and  Mg 
deposits. 

Specific  Gravity.  1020  average.  Taken  by  means  of  a 
urinometer.  This  is  a  small  hydrometer  graduated  for 
the  purpose  from  0  to  50,  the  last  figures  of  the  S.G. 
expressed    in    four   figures. 

Inorganic  Constituents. 
"Water.  Varies  according  to  activity  of  skin ;  the  S.G. 
indicates  its  proportion  to  total  solids.  A  rough  approxi- 
mate estimation  of  the  total  solids  for  urines  of  ordinary 
densities  can  be  made  by  the  Christison-Tyson  formula.  The 
quantity  of  solids  per  1,000  parts  urine  is  obtained  by  multi 
plying  the  last  two  figures  of  the  S.G.  expressed  in  four 
figures  of  urine  of  24  hours  by  2 '33.  Not  applicable  to 
urines   which    contain   sugar    or    albumin. 

Chlorides.      Chiefly   of     Na,     are    soluble,     do    not    deposit. 
In    24    hours,    10— 15g-     (Urea   to   chlorides,    2:1.) 

Test. — Take  one-quarter  tube  of  urine  ;  add  2  drops  HN03 
and  half  volume  of  AgN03  4pc-  solution  =  white  pp ; 
shake  thoroughly,  and  let  stand.  Deposit  normally 
about  one-quarter  volume  of  urine ;  the  HN03  keeps 
phosphates  in  solution. 
Increased    by    diet ;    diminished    in    fevers.     Not    clinically 

important. 


SULPHATES. — PHOSPHATES.  1  59 

Sulphates.    In    24    hours,  1*5    to    3g. 

There   are    two   classes    (in   proportion    10  :  1) — 

(a)  Ordinary    sulphates    of    K  and    Na    derived    from    proteids. 

of   food. 

(b)  Ethereal    sulphates.     The    potassium    sulphates    of   phenol, 

indoxyl,  skatoxyl,  derived   from   putrefactive   processes    in 
the  alimentary  canal. 

Recognition  : — 

Class    A    gives    a  pp    with    barium    chloride. 

B       „  „         „  „         „         „         after   boiling 

with   a    mineral    acid. 

Phosphates.    In    24    hours,    3-5 B   P,205. 

Three  kinds  of  salts  : —  Basic  (or  normal),  M3P04  ; 
monophosphates  (neutral),  M2HP04  ;  diphosphates  (acid), 
MH2P04. 

Clinically    they    are    of   importance    from    two    aspects  : — 

1.  Reaction    of  urine.     Acidity   largely  due   to  NaH2P04,  and 

alkalinity    to    Na3PO^. 

2.  Solubility.    Affects  the  production  of  sediments  and  calculi. 

(a)  Soluble   phosphates. — Na,   K,  NH3  do   not  deposit^  in   prop. 

(b)  Insoluble  phos2?hates. — Lime   and    magnesia  J      3:1 

The  earthy  phosphates  (b)  are  pp  in  an  alkaline  mediant, 
hence  they  appear  as  sediments  in  alkaline  urine.  In  ammo- 
niacal    urine    they    form    crystalline    deposits    as    follows  : — 

1.  Ammonio    magnesian    or    triple    phosphates,     (a)    Knife  rest 

or  coffin  lid  ;  (6)  feathery,  if  quickly  formed. 

2.  Stellar  calcium   phosphates  and  amorphous  deposit. 

On  heating  urine  a  turbidity  or  deposit  of  calcium 
phosphates  may  form,  due  to  the  transformation  of  the  acid 
into    the    basic    variety. 


160  HEALTHY    URINE. 

Recognition  : — 

1.  Turbid     urine     clears     on     adding     HISTOg     and     heating  = 

phosphates. 

2.  Two    drops    acetic    acid    and    drops    of    uranium    acetate — 

pp  =  P205    reaction. 

3.  Urine  +  half    volume     HISTOg    and    not  less   than    2    volumes 

of  molybdate  of  ammonia  =  yellow  pp  =  P205  reaction. 

4.  Pp  on  adding  NH3  (or  caustic  alkali)  =  phosphates  (earthy). 

^Q    Volumetric   estimation   of  P2Os   in   Urine. 

(a)  Standard    solution    uranium     nitrate     35-5 s    in    1     litre    of 

distilled    water.     lcc  =  -005s   P205. 

(b)  Acid  solution  of  sodium  acetate.     To  100  g  sodium  acetate 

add  100 cc  acetic  acid  33pc-  and  make  up  to  1  litre 
with  water.  To  liberate  all  the  P205  and  to  combine 
with  the  small  quantity  of  free  HISTOg  evolved  which  would 
dissolve  part  of  the  pp  (JSTeubauer). 

(c)  Solution  of  potassium  ferrocyanide  as  indicator.      It  yields 

a   brown   colour   with   the  uranium   acetate. 

Process : — 

1.  To  50 cc  of  the  urine  add  5  cc  of  (b)  and  maintain  at  100°  C. 

2.  In   a   burette  place   (a)   and   run    it    into    the    urine    slowly, 

and  test  a  drop  of  the  urine  from  time  to  time  with 
a    drop    of    the    indicator    on    a    white   plate. 

3.  Read  off  the  number  of  cubic  centimetres  of  the  SS  which 

used   and  calculate   the  amount   of    P205   as   follows : — 

P205  in  24 

CC  of  SS  used  x  0-005  x  CC  of  Urine  in  24  hours       , 
—  sb    hours    in 

50 

grammes. 


ORGANIC    CONSTITUENTS.  161 

Organic  Constituents.  17 

Urea  average  percentage  2pc-  (Blood  0-02  pc)  Quantity  in 
l'4  hours  about  30 g  depending  on  the  flesh  in  the  food. 
Occurs   in  animal  fluids,  not  in  muscle   (creatin). 

Properties : — 

1.  Crystallised  slowly,  forms   prisms;  quickly,  long  needles. 

2.  It  is  freely  soluble  in  water  and  absolute  alcohol,  but  insoluble 

in   ether  and   benzene. 

3.  On    heating    dry    to    130°  C.  it    melts,  giving  off   NH3  with 

the   formation   of  biuret   as   follows  : — 

Mo    co<:J 


2C0|";tt2  =  ^"NH  +NE 
\NH2     co< 


NH.2-co<^ 
Urea.        Biuret. 


3CO(NHo)2  =  C3H3N303  (cyanuric  acid)  +  3  NH3.  Cyanuric  acid 
solidifies  in  the  tube,  and  yields  on  further  heating  cyanic  acid 
which  volatilises,  no  residue  being  left  if  the  urea  is  pure. 

4.  It  combines  with  mineral  acids,  metallic  oxides,  and  salts 
to  form  compounds  in  which  the  molecule  of  urea  is 
united  to  one  or  more  of  the  reagents.     These  form  pps. 

.Recognition  of  the  solid  substance.  Use  commercial  urea  in 
crystals. 

1.  Place    some    crystals    in    a    dry   tube   and    heat,  they   melt 

(130°  C.)  with  the  odour  of  NH3.  Cool,  add  a  few  drops 
of  water   and   apply   the   biuret   reaction. 

2.  Dissolve  a  few  crystals  in   a   drop   of   water,   place  portions 

on  glass  slips.  Add  to  one  HN03  and  to  the  other  a 
saturated  solution  of. oxalic  acid.  Each  yields  a  crystalline 
mass  with  characteristic  crystals  of  nitrate  and  oxalate 
of  urea   respectively.     Examine   the   crystals. 


162  HEALTHY    URINE. 

3.  Schiff's  test.  Place  a  crystal  of  urea  in  a  porcelain  capsule, 
pour  upon  it  a  drop  of  concentrated  furfurol  solution 
in  water  and  add  a  drop  of  HC1,  a  play  of  colours 
changing  through  yellow,  green,  blue  to  violet,  and  in 
a  few  minutes  to  a  j^u^te-violet.  The  change  is  some- 
what slow,  the  purple  colour  being  the  best  marked. 
Old  furfurol  solutions  colour  red  on  the  addition  of 
HC1. 
To  a  solution  of  the  crystals  apply  the  following  tests  : — 

1.  Add  acetic  acid   and   a  few  drops  of  a  solution  of  mercuric 

nitrate — a    white  pp — (the   basis   of    Liebig's    volumetric 
method). 

2.  Add     a     solution    of      sodium     hypobromite  —  effervescence 

occurs. 

Quantitative    Estimation    of    Urea. 

Knop-Hihfner's  process  (or  some  modification  as  that  now 
given). — Depends  upon  the  decomposition  of  urea  by  a  hypo- 
bromite  or   hypo-chlorite.     Reaction  : — 

CO&  +  3£a>0  =  C02  +  SNaBr  +  2H„0  +  N,{EscaPes  anf 
^NH2       Br  2 %_s  -^is  measured. 

remain  in  the  solution. 

Theoretically  lg  urea  yields  0*46666 g  nitrogen.  The  volume 
of  this  at  0°  C.  and  760  mm  Hg  =  372-7 cc.  Practically 
354 cc  are  obtained  at  the  ordinary  temperature  (18°C.)  and 
pressure.  If  in  a  preliminary  trial  more  than  2pc-  is  found 
to    be   present,  the   urine    should    be   diluted. 

Process.  Do  this  first  with  a  2pc-  solution  of  urea  in 
water,  then  with    urine. 

1.  Preparation  of  sodium  hypobromite  solution  (Knop).  Dis- 
solve 100 g  NaOH  in  250 cc  distilled  water;  when  cool, 
add  25 cc  of  bromide  (in  the  open  air  or  a  draught 
closet). 


UKKAMKTKIi. 


L63 


? 


u 

—     ~~ . 

l"^"^  - 

-   ) 

D 


cc 

-50 


-40 


-30 


—  20 


— 10 


^ 


B  C  E  F 

Fig.  17.    Simple  Urea  Apparatus. 
2.  Apparatus1  : — 

Fill  tube  A  up  to  the  mark  (5CC)  with  urine  (or  urea 
solution),  and  place  it  in  B.  Into  B  place  25 cc  hypo- 
bromite  solution,  push  the  stoppers  firmly  into  B  and  C; 
the  tube  D  will  fill  and  water  should  drop  into  E. 
When  this  flow  ceases  empty  E,  replace  it  so  that  the 
point  of  D  is  near  the  bottom  of  E.  Now  tilt  B 
so  that  urine  and  reagent  mix  thoroughly ;  the  nitrogen 
froths  out  and  expels  an  equal  quantity  of  water 
from  C.  Allow  B  to  cool  four  minutes,  then  measure 
H20  in  E  with  F  =  cc  of  nitrogen  given  off.  Then 
since  Is  urea  yields  354 cc  nitrogen,  the  quantity  per 
cent,   is    found    thus  : — 


20  x  cc  1ST   obtained    from    5 cc    urine. 
354 


=  Urea  per  cent. 


irrhe  number  of  ureameters  which  have  been  devised  is  large.  The  majority 
have  their  gas  vessels  graduated  in  percentages  of  urea.  The  above  apparatus, 
which  can  be  easily  constructed  by  anyone,  is  sufficiently  accurate  when  the  nature 
of  the  process  is  taken  into  account. 


164  HEALTHY    URINE. 

From   which    again    the    quantity    in    24    hours    is    easily 
obtained. 

Note. — The  nitrogen  evolved  in  the  ahove  process  is  not  all  derived  from  urea 
in  urine,  but  comes  also  from  uric  acid  and  creatinine.  This  is  practically 
balanced  by  a  loss  of  N  from  the  urea,  which  remains  in  the  alkaline  solution  in 
the  form  of  nitrate,  and  partly  as  an  unknown  organic  compound  which  gives  off 
ammonia  when  distilled  with  alkali. 

Estimation  of  the  total  Nitrogen  in  Urine  by  Kjeldahl's 
process.       There  are  three  steps  in  the  process  : — 

A.  Incineration.  Namely,  decomposition  of  the  nitrogenous 
material  by  means  of  strong  sulphuric  acid,  whereby  ammonium 
sulphate  is   formed.     This  contains  all  the  nitrogen. 

B.  Distillation.  The  ammonia  is  liberated  by  the  addition  of 
an  excess  of  caustic  soda,  and  is  distilled  over  into  a  measured 
quantity   of   standard   acid,  a  part   of  which   it   will   neutralise. 

C.  Titration.  The  unneutral ised  portion  is  measured  with 
standard  alkali,  and  thereby  the  quantity  which  has  been 
neutralised  by  the  ammonia  becomes  known.  From  this  the 
quantity   of  nitrogen   is   readily   calculated. 

Reagents   required  : — 

1.  Sulphuric  acid,  strong   pure,   15  g- 

2.  Potassium   sulphate,  dry,   10  g- 

3.  Sulphate  of  copper,  dry,  0*5  g- 

4.  Caustic  soda  solution,  25 pc-,  boiled  with  zinc  shavings 
to   free   it  from   nitrates. 

N  .  N 

5.  — — -  NaOH  solution.    Dilute  stock  — —  solution  with 

9  volumes  of  distilled  water. 

N  N 

6.  — -H2S04.     Dilute  stock— —solution    with    9    volumes 

of  distilled  water. 

7.  Methyl  orange.     1  :  1000  solution. 

8.  Powdered  talc.      Half  a  teaspoonful. 


KJELDAHLS    PROCESS. 


165 


Process  : — A.  Incineration.  10  cc  of  urine  are  measured  from 
a  burette  into  a  hard  glass  flask,  to  this  add  15 cc  sulphuric 
acid,  10 g-  potassium  sulphate  and  0'5  g-  of  sulphate  of  copper 
crystals.  K2S04  raises  the 
boiling  point,  and  CuS04 
helps  oxidation.  Close  the 
mouth  with  a  loose  balloon 
stopper,  and  support  the 
flask  on  wire  gauze  with 
the  neck  in  an  inclined 
position.  Raise  the  heat 
gradually  and  boil  until 
the  blackened  fluid  be- 
comes quite  clear  (green 
from  the  CuSOJ.  This 
step  must  be  carried  out 
in  a  draught  chamber  on 
account  of  the  sulphurous 
fumes  which  are  given  off. 
When  quite  cool  add 
slowly  50  cc  distilled  water. 
As  this  is  attended  by 
much  heating  cool  again. 
Add  a  few  drops  of  methyl 
orange  and  half  a  tea- 
spoonful  of  talc.  Next 
add  caustic  soda  to  liber- 
ate the  ammonia,  pouring  it  along  the  side  of  the  vessel  until 
the  reaction  is  nearly  alkaline,  cool,  render  alkaline  by  a  further 
addition  of  NaOH,  and  connect  at  once  with  the  distilling 
apparatus,  which   has  meanwhile   been  prepared. 

•I  am  indebted  to  ray  colleague  Prof.  Proctor's  work,  "Leather  Industries," 
for  the  method  of  distillation  without  a  condenser,  and  to  his  assistant,  Dr. 
Guthrie,  for  the  more  recent  modifications  in  the  apparatus. 


Fig.  18.     Apparatus  for  Kjeldahl's  Process.1 

A  Hard  Jena  or  Bohemian  glass.  B  Bulb 
to  prevent  spurting  over  of  alkali  dui-ing  distil- 
lation. C  Erlenmeyer  flask  surrounded  by  a 
cooling  vessel  containing  water.  D  Tube 
containing  glass  beads  or  broken  glass.  E  To 
arrest  regurgitation.  E  and  B  are  made  out 
of  pipettes. 


166  HEALTHY    URINE. 

B.  Distillation.       Into    the     Erlenmeyer    receiving    flask   (C) 

N 
measure    from    a    burette    80 cc    of  —    (decinormal)    sulphuric 

acid.  This  is  in  excess  of  what  will  be  required  to  combine 
with  all  the  ammonia  }7ielded.  Pour  this  in  through  the  side 
tube  (D)  to  wet  the  glass  beads  as  a  precaution  to  catch  any 
ammonia  that  might  escape  the  sulphuric  acid.  There  is  little 
danger  of  this,  however.  Add  a  few  drops  of  methyl  orange, 
which  would   indicate   the   neutralisation  of  all   the   acid   when 

more   ^—  ELS04  would  have   to  be  added.     The  delivery  tube 

(E)  must  dip  a  little  below  the  fluid  in  (C)  during  the  first 
part  of  the  operation,  when  most  of  the  ammonia  conies  over. 
The  great  affinity  of  the  sulphuric  acid  for  ammonia  renders 
loss  unlikely.  Check  violent  bumping  in  (A)  by  reducing  the 
gas  flame.  The  bulb  (E)  saves  the  contents  of  (C)  from 
regurgitating  into  (A).  Should  the  fluid  regurgitate  into  (E) 
and  ammonia  be  present,  it  will  turn  the  methyl  orange  yellow 
and  serve  as  a  useful  indication  that  there  is  still  ammonia 
coming  off.  (C)  must  be  kept  cool  by  changing  the  water 
from  time  to  time.  As  soon  as  (A)  begins  to  bump  vigorously 
the  ammonia  may  be  assumed  to  have  been  all  expelled 
(twenty   minutes). 

The  flask  (A)  should  now  be  detached. 

(C)  Titration.     Cool  (C),  wash  out  (D)  into  (C)  with   10ccof 
distilled  water,   and   then   titrate   the   uncombined   acid    in   the 

N  N 

latter  with   — -  NaOH.     The  — ■    NaOH     is     run    in    from    a 

burette  until  the  methyl  orange  turns  yellow,  the  last  quantity 
required   being   added   drop   by   drop. 

Calculation  : — 

l'c  —  NaOH  =  1«  ^H2S04  =  0-001 7gNH..  =  0-0014«N. 
10  10      ~ 


CREATININ. UIUC    ACID.  167 

N 
From   the   quantity  of     -     H.,S04    originally    measured    into 

X 
the  receiver  (C)  subtract  the  cc—  NaOH   used    to   neutralise 

the  uncombined  acid,  and  multiply  the  difference  by  0-0014, 
which  will  give  the  grammes  of  nitrogen  contained  in  10 cc 
of  urine.  The  percentage  will  be  obtained  by  multiplying 
by    10. 

Creatinin.     lg  or  over    in    24    hours.     Is   derived    from    the   19 
fleshy    part    of   food.     It    comes    next  in  importance    quantita- 
tively after    urea.     Is    freely    soluble    in    water    and    has    an 
alkaline    reaction. 

1.  It   reduces    copper   oxide,    hence    may    be    taken    for    small 

quantities  of  sugar.  The  reduced  copper  collects  at 
the   bottom    of   the    tube.     Try  normal   urine. 

2.  WeyVs    reaction.     Add    to    a    quarter    tube    of    urine    a  very 

dilute  solution  of  sodium  nitroprusside,  and  then  drop 
by  drop  a  dilute  solution  of  NaOH,  a  ruby  red  colour 
appears  which  lasts  only  a  few  minutes  and  passes 
into  a  clear  straw  colour.  Add  some  acetic  acid,  the 
ruby  colour  rapidly  decolourises.  Compare  with  the 
reaction    for    acetone. 

Uric  Acid.    0-5    a   little   more   in    24   hours. 
Properties  : — 

1.  Its    solubility  is   very  low— 1    to   14,000    in    cold,  and   1   to 

1,800  in  boiling  water.  Most  important  from  a  clinical 
point  of  view,  as  consequently  it  readily  appears  as  a 
sediment  in  the  urine. 

2.  It    crystallises    ordinarily   in    urine    in    the    form    of    whet- 

stone -  shaped  crystals,  coloured  brownish  -  red  by 
entangled  urinary  pigment  (uroerythrin).  When  crystal- 
lised   pure    it    forms    rhombic    prisms. 


168  HEALTHY    URINE. 

3.  Dissolves    in    KOH,    potassium   urate    being    formed,    it    is 
re-pp   by   the   addition    of    5pc-  HC1   as    uric   acid.     This 
addition  of    HC1    to   urine  causes    it   to  separate   out   in 
the   form    (2). 
Tests1:— 

1.  Murexide  test.     To  a  few  drops  of  urine  add  a  little  HN03 

evaporate  on  a  porcelain  dish  without  charring,  cool  and 
add  NK — a  purple  colour  or  with  KHO  a  violet — 
murexide. 

2.  Schijfs  test.     Dissolve  some  uric  acid   crystals1  provided  for 

you  in  sodium  carbonate  solution,  drop  on  a  filter  paper 
moistened  with  AgN"Oa  a  black  stain  of  reduced  silver 
results. 

3.  Solutions  of  uric  acid  or  acid  urates   reduce  alkaline   copper 

solutions.  Use  potassium  urate  obtained  by  dissolving 
serpent's  excrement  in  KOH.  Part  of  the  Cu20  formed 
unites  with  any  undecomposed  uric  acid  to  an  "insoluble 
pp  of  cuprous  urate. 

4.  To    some    urine   add    5pc-  HC1,  let   it   stand    for    24    hours. 

Brown  whetstone-shaped  crystals  separate  out  as  a  surface 
scum  and  as  a  deposit.     Try  the  tests  on  these. 

Urates.      Uric  acid  is  a  dibasic  acid  (H2U)  and  forms  three 
kinds  of  salts. 

(a)  Normal  urates  (M2U).     These  do  no  occur  in  the  body  and 

are  laboratory  products  only. 

(b)  Acid  or  Bi-Urates  (MHU)  they  occur  as  gouty  concretions. 

(c)  Quadriurates  ■[  \t  tt  [  These  according  to   Bence  Jones  and 

Sir  W.  Roberts  are  the  physiological  salts  of  uric  acid. 

They  tend  to  break  up  in  the  presence  of  water  into 
acid  urates  and  a  molecule  of  additional  uric  acid.     This 

1  Serpent's  excrement  which  consists  of  nearly  pure  urate  of  ammonia  may  be  used 
with  advantage  for  performing  these  tests. 


URATES. — OXALATE. MUCIN.  169 

is  ordinarily  hindered  in  urine  by  the  presence  of  phos- 
phates, chlorides  and  pigments.  The  conditions  which 
accelerate  the  change,  the  converse  retarding,  are  (1) 
acidity,  (2)  poverty  of  mineral  salts,  (3)  little  pigment, 
(4)  a  high  percentage  of  uric  acid. 

Demonstration  of  Quadriurates.  Place  a  slight  scraping  of 
serpent's  excrement  upon  a  glass  slip,  press  it  into  a  fine 
powder  with  the  blade  of  a  knife  and  shake  off  all  that 
does  not  adhere  to  the  glass.  Cover  dry  and  examine  under 
a  power  of  300  diams.,  observe  the  small  globular  masses 
of  quadriurate  of  ammonia  which  alone  are  present.  Let 
water  flow  between  the  glasses  and  recognise  the  almost 
instantaneous  appearance  of  a  crop  of  short  square-ended 
needles  of  uric  acid,  which  grow  into  fine  parallel  sided 
colourless  crystals  or  thin  fusiform  plates.  Ammonium 
urate  remains  as  granules. 
Acid  Urates    are     far    more    soluble    (at    least     10    times) 

than  uric  acid ;    nevertheless,  they  are    the   commonest  pps  in 

urine. 

Their  deposition   is  favoured  by  (1)  an  acid   reaction,  (2)  low 

temperature,    (3)   little    water,    (4)   ammonium    urate    occurs    in 

alkaline   urine. 

A  pp  of  urates  in  urine  is  coloured  fawn  to  brick  dust  red, 

depending  upon  the  quantity  of  pigment  involved   (uroerythrin). 
These  pps  dissolve  on  heating,  and  so  differ  from  phosphates 

which   require   an   acid. 

Oxalate  of  Lime.  1 g  in  24  hours.  Is  kept  in  solution  in 
the   urine  by   acid    sodium  phosphate. 

It  appears  as  a  sediment  after  eating  rhubarb,  cabbage,  &c, 
as  crystals  which  are  regular  octohedra,  brilliant,  and  colourless. 

Mucin.  Normally  present  in  small  quantity,  collects  on 
standing  as  a  faint  cloud   at   the  .bottom  of  the  vessel. 


c ... 


CHAPTER   XXVI. 

ABNORMAL    URINE. 

Colour.  If  very  pale,  probably  due  to  unusually  large  quantity 
of  water — polyuria.  If  high-coloured,  suspect  proteids ;  if  red, 
brownish,  chocolate,  or  deeper  coloured,  some  form  of  blood 
pigment ;  if  orange  reddish,  or  darker,  with  a  greenish  tint  at 
the   top,  bile   pigments. 

S.G.  and  quantity.  Remember  these  are  interdependent. 
A   high   S.G.  with   large   quantity   indicates   sugar. 

Albuminuria.  Any  of  the  following  may  occur,  serum- 
albumin  and  globulin  usually  together.  Their  separate  recogni- 
tion is  at  present  of  no  clinical  importance.  Blood  .pigments 
will   give   proteid   reactions. 

Tests : — Those  for  proteids  generally. 

1.  Heller's  contact  method  should  be  tried  first.  Remember  that 
an  old  iron  spoon,  a  tallow  candle,  and  some  vinegar 
will  enable  you  to  perform  the  recognition  of  a  coagulable 
proteid   under   difficulties. 

Quantitative  Estimation.-  By  Esbach's  Albuminometor.  Albu- 
min   and   globulin. 

1.  Reagent: — 10 g  picric  acid  and   20 g  citric  acid   to   1   litre  of 

water. 

2.  Urine   must  be  acid,  add  acetic  acid  if   required.     S.G.  must 

be  lowered  to  1008  by  dilution.  The  process  is  most 
accurate  when  4 K  of  albumin  per  litre  are  present.  The 
temperature  has  a  marked  influence.  The  colder  the 
urine   the   bulkier   will    the  pp  be. 


ALBUMOSURIA.  —  HJEMOGLOBINURIA.  171 

3.  Measuring   tube. 

Process. — Fill  the  measuring  tube  with  urine  to  the  mark  U, 
add  the  reagent  to  R,  cork,  and  invert  10  to  12  times 
(no  shaking).  Set  the  tube  upright  in  a  stand  for  12 
hours  and  read  off  the  percentage  from  the  graduations, 
which  give    it  in   grammes   per  litre. 

The  separate  recognition  of  globulins  and  albumins  is  of  no 
importance  clinically.  That  globulin  is  present  may  be 
demonstrated  : — 

1.  By   dropping  urine   into  a   large  quantity   of  water — a  cloud 

forms. 

2.  ^Neutralise  urine  carefully,   filter   off   any  pp  of    phosphates, 

then  add  one-half  its  volume  of  a  sat.  solution  Am.2S04, 
the  pp  indicates  globulin.  Filter.  Albumin  can  then  be 
demonstrated  in   the  usual  way  in  the   filtrate. 

Albumosuria  and  peptonuria.  See  tests  already  given 
under   the   respective   substances. 

The  most  convenient  method  is  to  saturate  some  of  the 
urine  with  JSTaCl,  add  drops  of  acetic  acid,  boil  in  a  beaker, 
filter  hot.  The  filtrate  gives  on  cooling  a  pp  of  mixed 
albumoses.  If  there  be  no  pp,  a  pink  Biuret  reaction  shows 
pepton. 

Haemoglobinuria.  Blood  in  urine  may  appear  as  Hb02, 
Met-Hb,  Acid   H-tin,  Hasmatoporphyrin. 

Recognise  these  by  the  tests  already  given  and  especially  by 
the    following  : — 

1.  Heller's   test.     To   the  urine  add  one-eighth  volume  NaOH, 

boil ;  a  pp  of  phosphates  coloured  red  by  hsematin 
results. 

2.  Guaiacum   test,   pg.    \'6~ . 


172  ABNORMAL    URINE. 

3.  Search   for  blood   corpuscles. 

4.  Spectroscope. 

Mucin,  Is  increased  in  cystitis  and  may  be  derived  from 
the   vagina.      Tests  : — 

1.  Dilute  the  urine,  if   the   S.G.  is   high   to  prevent   the   salts 

keeping  the   mucin   in  solution,  with   2  volumes  of  water 
and  add  acetic   acid  pp. 

2.  If  there  be  a  heavy  deposit  in  the    urine    collect    some   of 

it    with    a   pipette,  place    in   a    tube    and    add    drops    of 
ISTaOH,  the  mucin  will  lose  its  viscidity  and  become  fluid. 

Pus.  Donne's  test.  Collect  some  of  the  deposit  with  a  pipette 
and  add  to  it  some  IsTaOH  solution.  It  thickens  and  the  fluid 
moves  sluggishly  in  the  tube  on  shaking,  alkali  albumin 
being  formed. 

Cystinuria.  The  crystals  of  cystin  are  found  in  urine  in 
rheumatism.  It  may  occur  as  a  constant  constituent  to  the 
extent   of   0-5 g  in    24   hours,   but   is   usually   abnormal. 

1.  It  is   insoluble  in   water. 

2.  The   crystals   are   six-sided  plates. 

21  Glycosuria,  sugar  in  urine,  saccharine  urine.  A  very 
small  quantity  may  be  normally  present  in  urine.  The 
quantity  is  large  in  diabetes  mellitus.  The  appearance  of 
more  than  a  mere  trace  of  a  reducing  substance  is  clinically 
important.  The  presence  of  dextrose  is  indicated  by  the 
usual   tests. 

Tests.     Remove  proteids   as   they  interfere. 

(a)    With   Copper  Salts. 

Trommels  test  is  the  type.  Make  urine  strongly  alkaline  with 
KOH  (or  NaOH)  add,  with  care,  by  drops  solution 
CuS04  (l1,c).      On   heating  a  yellow  or  red  pp  of  cuprous 


GLYCOSURIA.  173 

oxide  (Cu20)  occurs,  which,  if  well  marked^  and  abundant, 
indicates  either  sugar  or  glycuronic  acid.  The  latter  is 
not  very  common,   but  should   be  remembered. 

Note. — In  this  test  if  performed  with  H20,  the  hydrated  copper 
oxide  produced  by  the  alkali  on  CuSo^  is  insoluble,  and  heated  to  100° C. 
falls  as  a  black  pp.  Ordinary  urine  differs  from  H.20  in  keeping  the 
hydrated  oxide  in  solution,  and  yields  a  slight  reaction  due  to  uric  acid, 
hippuric  acid,  creatinin,  alcapton,  albumin,  nucleo  albumin,  bile  pigments. 
These  may  veil  the  reaction  due  to  the  minute  trace  of  sugar  normally 
present.    Fehling's  solution  and  BarfoecVs  reagent  give  the  same  reaction. 

(b)  Bismuth  Salt. — Nylander's  test.     To  10  volumes  urine  add 

1  volume  of  the  following  solution :  2 e  basic  nitrate  of 
bismuth,  4  s  Rochelle  salt,  100 cc  of  a  solution  containing 
10-33g  NaOH.  Boil  5  to  10  minutes.  A  black  pp  forms 
(said  to  show  as  little  as  -04  p,c-  sugar).  Albumin  gives 
it  also,  6 pc-  a  red  brown  pp),  and   1-2  pc-  a  black  pp. 

Pure  peptone  does  not  give  the  reaction    (Le  Nobel). 

It  is  not  reduced   by  uric  acid,   creatinin,  alcapton. 

(c)  Phenyl   hydrazin    test. — Place  a  small    quantity  of    the  dry 

substance  (knife  point),  phenyl  hydrazin  hydrochlorate, 
and  two  or  three  of  acetate  of  soda  in  half  a  tube  of 
urine.  If  they  do  not  dissolve,  add  a  little  water.  Boil 
30  minutes,  then  cool.  Yellow  crystals  deposit.  Under 
the  microscope  they  appear  as  yellow  needles. 

Quantitative    Estimation. 

Precautions. — Remove  any  proteids  that  may  present  by 
acidulating,  boiling,  and  filtering. 

In  performing  a  volumetric  estimation  with  Fehling  or  Pavy's 
modification  it  should  be  noted  that  the  suboxide  formed 
may  be  re-oxidised  by  the  oxygen  of  the  atmosphere  and 
that  in  consequence  a  partial  return  of  the  blue  colour  may 
be    occasioned    even    during    the    performance    of    the    process. 


174  ABNORMAL    URINE. 

It  is  therefore  necessary  to  proceed  quickly  and  to  delay  as 
little  as  possible  over  the  earlier  part  of  the  operation,  slowing 
towards  the  end.  Three  estimations  should  be  performed. 
The  first  as  rapidly  as  possible  by  running  the  sugar  solution 
continuously  into  the  boiling  Fehling  until  discoloration  is 
complete. 

From  this  preliminary  trial  is  ascertained  approximately 
how  much  sugar  solution  is  required  to  reduce  all  the  copper. 
In  the  next  run  this  amount  less  2  or  3CC  into  the  Fehling, 
care  being  taking  not  to  lower  the  temperature  of  the 
Fehling  below  boiling  by  a  too  rapid  addition.  With  the 
third    trial    a    close    approximation    should    be    attained. 

Should  the  sugar  solution  be  so  strong  that  only  a  few  cc 
of  it  reduces  all  the  copper,  then  further  dilution,  to  say 
1  in  40,   is  necessary. 

Fehling 's  solution.     10cc  =  O05g  dextrose. 

1.  Dilute  the  urine  with  19  volumes  H20,   and  place  it  in 

a   burette    ( =  1    in    20    solution). 

2.  Dilute  10cc  Fehling  +  40 cc  H20,  and  place  in  a  porce- 
lain   capsule.     Keep   it   boiling. 

3.  Run  (1)  into  (2),  until  all  Cu20  is  pp  and  the  blue 
colour  is  gone.  To  determine  this,  tilt  the  capsule  to  utilise 
the    white   back    ground. 

4.  Read  off  the  number  of  cc,  dilute  urine  used,  then 
20  :  cc    dilute    urine    used  :  :  1  :  x  =  cc    actual    urine    used. 

x   contains    '05  s    sugar. 

rv     n     1    ^                   *           100  x -05 
To  find   the  percentage    =  y. 

x 

y  x  cc    urine    in    24    hours     ^        ...       .      0/1     , 
J- =  Quantity    in    24    hours. 

1 00  J 


PAW. — FEHLIN6.  17  ~J 

Pavys   modification    of  Fehlingt'a   process. 

Principle: — Cuprous  oxide  is  dissolved  by  NH3.  A  sufficient 
quantity  of  NH..  is  added  to  Fehling  to  dissolve  the  CuX) 
formed,  the  disappearance  of  the  blue  colour  can  thus  be 
better   seen.     The    end   of    the    reaction    is    more   distinct. 

S.S.  120cc  Fehling  +  400  CCNHS  (0-88) +  H20  to  1  litre.  10cc  = 
lcc  Fehling  =  0*005  g  sugar.  The  solution  keeps  well  in  properly 
stoppered  bottles.  It  can  be  obtained  in  sealed  tubes  contain- 
ing   10cc. 

Process  : — 

1.  Place    10cc    Pavy-Fehling    in    a    flask    fitted   with    a    good 

cork  through  which  two  tubes  pass.  The  one  is  short 
and  is  joined  to  the  nozzle  of  the  burette  by  a  rubber 
junction,  the  other  is  bent  to  an  angle  outside  the 
cork    and    leads    into    the    air. 

2.  The   burette,    as    in    the    former    case,    contains    a    1    in    20 

dilution   of   the   fluid    under   investigation. 

3.  Boil    the    standard    solution,   and   whilst  it  boils   run  in  the 

sugary    fluid    from    the    burette    with     the     precautions 
mentioned.      Note    the    disappearance    of    the    colour,    to 
assist   which    hold   a  piece    of    white    paper   or    a    white 
tile    behind    the    flask. 
Make   a   second   and    third    estimation   as    before. 

Fermentation  Process.  Important.  Glucose  ferments  ; 
Glycuronic  acid  does  not.  To  perform  the  .estimation  with 
the  greatest  degree  of  accuracy,  good  hydrometers  are  required, 
and  should  be  employed  at  the  temperature  for  which  they 
are    graduated. 

Process  : — 

1.  Take    S.G.   of   urine,   filter  it,   and   place    100 cc  in  a  flask. 

2.  Add   some   yeast   of   the   size   of   a   pea,    mix  thoroughly. 


176  ABNORMAL    URINE. 

3.  Stand   the   mixture   in   a  warm    place    for    24    to    48   hours. 

To  prevent  evaporation,  it  is  desirable  to  have  a  tube 
trapped   with   water,    passing    through    the    cork. 

4.  Filter     quickly,     take     S.G.    at     the    same     temperature     as 

before. 

5.  Multiply   the    difference    in    S.G.     by    230,    this    gives    the 

percentage.  Example— 1  -040  -  1  -008  =  -032  x  230  =  7 -36 13C- 
Or  each  degree  of  difference  =  1  grain  per  fluid  ounce. 
(Sir  W.  Roberts.) 

Examine  Einhorrfs  Fermentation  Saccharometer  and  Dr.  G. 
Johnson 's  Picro-saccharometer. 

Acetone  occurs  in  the  urine  at  times  and  is  often  associated 
with  glycosuria,  pg.  128. 

Bile  in  Urine.  Icteric  Urine.  Note  the  colour — orange  to 
greenish-brown,  or   even    to  dark   porter  colour. 

Tests  : — Perform  the  tests  for  pigments  and  bile  acids,  pg.  128. 
The  test  for  acids  often  fails  on  account  of  their  small  quantity. 


177 


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LIST    OF    REAGENTS. 

Acid,  Acetic,  commercial,  and  5p-g  solution. 

,,     Hydrochloric,  concentrated  and  0'2p-c-  solution. 

,,     Nitric,  {a)  concentrated,  and  (6)  containing  nitrous  acid. 

N 
,,     Sulphuric,  concentrated,   10p-c-  and  ~r  solution. 

Alcohol,  absolute  and  methylated. 
Ammonium  Hydrate  (S.G.  0'88). 

,,  Molybdate,  10p-c-  solution. 

„  Oxalate,  10p-c-  solution. 

,,  Sulphate,  crystals. 

BarfoecVs  reagent,  copper  acetate  1,  water  *15.  To  200  ■'  add  5CC 
commercial  acetic  acid. 

Barium  chloride,  10 pc-  solution. 

Calcium         ,,        2p-c-  solution. 

Congo  red. 

Copper  acetate,  crystals. 

,,       sulphate,  crystals  and  4p-c-  solution. 

Ether,  methylated. 

Fehling  solution,  (a)  of  pure  CuS04  crystals,  36*448  distilled  water 
.")00 cc.  {b)  Rochelle  salt  (potassium  sodium  tartrate)  173 s,  sodium 
hydrate  60  g,  dissolve  separately  in  distilled  Avater,  mix  together,  and 
make  up  to  500 cc.  (a)  and  (b)  combined  in  equal  quantities  for  the 
standard  solution,    10 cc   of   which  =  50  m&  of  glucose. 

Ferric  chloride,  101,c-  dilution  of  liquor  fortior  B.P. 

Furfurol. 

Guaiacum  resin. 

Hydrogen  peroxide. 

1 1, 'line  solution.     Iodine  1,  potassium  iodide  2,  water  300  parts. 

Lead  acetate    bade. 


LIST     OF     REAGENTS. 


179 


Litmus,  solution  and  paper. 
Magnesium  sulphate,  crystals. 
Mercuric  chloride,  aqueous  solution. 

Methyl  orange. 

MiUon's  reagent.      Dissolve    mercury    in   an   equal   weight   of    concen- 
trated   nitric   acid  :    heat    to  solution  ;    add   2    volumes   water  ;    decant 

next   day. 

Phenol. 

Phenyl  hydrazin. 

Picric  acid  saturated  solution. 

Potassium  Ferricyanide,   10p-c-  solution. 

,,  Ftrrocyanide,      ,,  ,, 

Hydrate, 

,,  Sulphate. 

Silver  nitrate.  4p-c-  solution  in  distilled  water. 
Sodium  acetate,  crystals. 

,,        carbonate,  10pc-  solution. 

,,        chloride,  crystals  and  10  p°-  solution. 

N 
,,         hydrate,   10p-c-  and  ~r~  solution. 

Sodium  nitro-prusside. 

Sulphur,  flowers  of. 

Tannic  Acid. 

Uranium  Nitrate, 


PART    III. 

EXPERIMENTAL    SECTION. 

Students  work  in  pairs,  a  sub-division  of  labour  which  mater/"?/// 
facilitates  the  performance  of  the  various  experiments. 

Each  student  is  required  to  bring-  the  following- :— One  pair  of 
ordinary  dissecting  and  one  pair  of  fine  pointed  small  scissors,  cutting  well 
at  the  points.  One  pair  of  ordinary  dissecting  forceps  and  one  small  fine 
pointed  pair.  They  must  grasp  well  at  the  points  and  be  perfectly  clean. 
A  small  scalpel.  One  metal  and  one  glass  seeker.  Ordinary  pins.  Half 
a  dozen  S  hooks.     Some  fine  linen  thread  and  fine  sewing  silk. 


Fig.  19.     Glass  seeker,  with  ends  differently  bent. 

Each  work  table  is  supplied  with  the  following,  which  are 
placed  on  the  shelf  below  it  : — An  induction  machine.  Two  Leclanche 
cells  (quart  size).  Three  electrical  keys  (switch  pattern).  A  porcelain 
bowl  and  plate.  Wires  for  electrical  connections.  A  stick  for  killing- 
frogs.     A   duster.     A   bottle    of   normal   saline. 

Each  table  is  provided,  as  occasion  demands,  with : — A  record- 
ing cyclinder.  A  general  stand  with  a  muscle  chamber,  frog  heart 
recorder   and   time   marker.      A  variable  spring.      Daniell  cells. 

Note. — The  chapter  on  Electrical  Considerations  should  be  read 
carefully    beforehand. 


fet 


CHAPTER    XXVII. 

ELECTRICAL      CONSIDERATIONS. 

/■."  trieity  comes  under  consideration  in   Physiology  as  follows  :- 

1.  As  a  convenient  stimulus  for  muscles  and  nerves  in  the  form  of  induction 
currents. 

•_'.  As  a  direct  battery  current  for  the  production  of  polarisation  in  nerves  and 
exceptionally  in  this      orm  as  a  stimulus. 

:;.  As  a  direct  battery  current  for  actuating  mechanical  contrivances,  such  as 
time  markers,  &c,  the  basis  of  which  is  the  electromagnet. 

4.  The  passage  of  a  current  through  a  conductor  in  the  proximity  of  a  magnetic 
needle  causes  the  needle  to  move.  This  principle  is  made  use  of  in  the  galvanometer 
and  by  means  of  it  the  currents  yielded  by  living  tissues  can  be  recognised  and 
measured. 

Remember  .—Electromotive  force  (E.M.F.)  is  the  force  which  tends  to  move 
electricity  from  a  point  of  higher  to  one  of  lower  potential.  The  unit  of  E.M.F.  is 
the  volt,  and  is  therefore  the  measure  of  electrical  pressure.  One  volt  will  move  a 
quantity  of  1  coulomb  of  electricity  through  a  resistance  of  1  ohm  in  1  second, 
thereby  producing  a   current   of  1  ampere.      This  relationship  is  expressed   by  the 

formula  C=- 


B 

Current  (amperes)  = 


Total  E.M.F.  (in  volts). 
Total  Resistance  (in  ohms). 


SIMPLE 


CELL 


DANIELL 


— 

|  + 

z 

c 

CELL 


mm 

1 

1+ 

1 

1 

z 
+ 

1 

1 
H2<Cu-» 
so4  ,S0A 

"*"         1 
1 

1 

ci 

1 
1 
1 

1 

POLARISATION 


DEPOLARISAT10N 


Fig.  20.  The  tilm  of  H,  which  produces  polarisation,  is  shown  as  a  row  of  dots 
on  the  C  plate  of  the  simple  cell. 

The  galvanic  cell  consists  of  two  plates  or  elements,  commonly 
the  one  of  zinc  and  the  other  of  carbon  or  copper  immersed  in  a 
sensitising  fluid  (l(n,c-  H2S04).  The  action  of  the  cell  depends  upon 
the  solvent  action  of  the  acid  upon  the  zinc,  zinc  sulphate  being  formed. 
At  the  same  time  H  is  liberated.      This   transaction  is   pictured   in   the 


184  ELECTRICAL    CONSIDERATIONS. 

diagram  of  the  simple  cell  (Fig.  20).  The  immersed  portion  of  the  zinc 
is  the  positive  plate,  whilst  the  projecting  part  is  the  negative  pole 
(Kathode). 

The  current  flows  from  the  zinc  to  the  C  plate  in  the  fluid,  and 
circulates  from  the  positive  pole  (Anode)  to  the  zinc  through  the 
external  connection.  The  hydrogen  which  is  liberated  appears  on  the 
negative  C  element,  where  it  forms  a  film  which  is  not  only  non- 
conducting but  is  a  strongly  electro-positive  as  well.  This  has  an 
important  bearing  upon  the  working  of  the  cell,  as  there  is  an  increased 
resistance  offered  to  the  passage  of  the  current  by  the  film  of  hydrogen 
bubbles  on  the  negative  plate,  which,  at  the  same  time,  exerts  an 
electromotive  force  in  a  direction  contrary  to  that  of  the  cell,  and 
both  taken  together  ultimately  render  the  cell  inoperative.  This  is 
termed  polarisation.  A  corresponding  action  takes  place  between  metal 
terminals  (Electrodes)  and  the  animal  tissues  with  which  they  may  be 
in   contact  when   a   current   passes   through    them. 

Amalgamation  of  the  zinc  plate.  Pure  zinc  is  not  attacked  by 
the  acid.  Owing,  however,  to  the  presence  of  iron,  arsenic,  &c. ,  local 
circuits  on  the  surface  of  the  zinc  come  into  action  and  the  metal  is 
eaten  into  holes.  To  remedy  this  the  zinc  is  amalgamated  with 
mercury.  The  plate  is  first  cleaned  with  1  in  10  sulphuric  acid  in 
water  until  bubbles  of  gas  are  given  off,  and  is  then  rubbed  over  with 
mercury,  using  a  stiff  nail  brush  for  the  purpose,  so  as  to  produce  an 
even  covering,  and  to  remove  superfluous  mercury.  The  impurities 
are  thus  detached  from  the  zinc  and  a  more  nearly  pure  zinc  surface 
is  offered  to  the  acid,  and  local  action  is  largely  diminished.  Singing, 
i.e.,  the  formation  of  bubbles  should  be  at  a  minimum  when  the  circuit 
is  open. 

Galvanic  cells  differ  in  character  in  regard  to  roltage,  constancy  of 
action,  and  internal  resistance. 

Tlit  voltage  or  E.M.F.  of  a  cell  depends  upon  the  nature  of  its 
constituent  elements. 

The  internal  resistance  depends  upon  (a)  the  area  of  the  plates, 
(b)  the  distance  at  which  they  are  placed  from  each  other,  and  (c)  the 
resistance   of   the    sensitising   fluid. 

The  constancy  of  action  depends  upon  the  completeness  with  which 
polarisation  is  abolished. 

The  Daniell  cell.  This  is  the  standard  cell  used  for  physiological 
work.  The  zinc,  with  its  acid,  is  separated  by  a  porous  septum  from 
the  copper,  which  is  placed  in  a  saturated  solution  of  copper  sulphate 
(the    depolarising    agent).       The     hydrogen    liberated     unites    with    the 


GALVANIC    CELLS.  L85 

sulph-ion  from  the  copper  sulphate,  and  copper  is  deposited  upon  tin- 
copper  plate.  The  action  of  this  cell  remains  constant  as  long  as 
there  is  copper  sulphate  present  to  be  decomposed.  A  store  of  the 
salt  is  kept  in  the  solution  to  make  up  for  loss.  Voltage  1  *9, 
internal   resistance   0*5   ohm   approximately. 

Leclanche  cell.  In  this  the  acid  is  replaced  by  sal  ammoniac, 
the  solution  of  the  zinc  yields  chloride  of  zinc  and  ammonia. 
Ammonia  and  hydrogen  gas  are  liberated  at  the  carbon.  Depolari- 
sation  is  effected  by  means  of  manganese  dioxide,  which  is  packed 
round  the  carbon  plate  mixed  with  granulated  carbon.  Mn02  yields 
oxygen  slowly,  this  combines  with  the  hydrogen  to  form  water.  The 
hydrogen  collects  faster  than  it  is  removed,  and  the  cell  polarise.^. 
If  the  circuit  be  open  for  a  short  time,  the  cell  depolarises ;  it  is 
therefore  most  useful  for  open  circuit  work,  i.e.,  work  in  which  the 
circuit  is  closed  for  a  brief  time  only.  Voltage  1*5,  internal  resistance 
about    1    ohm. 

This  battery  is  mostly  employed  for  actuating  induction  machines 
and  time  markers,  and  works  well  provided  the  period  of  close 
circuit  is  shorter  than  that  of  open  circuit.  Most  "dry"  cells  are 
modified   Leclanche   cells. 

Other  cells  used  less  frequently  in  physiological  work. 

The  Gpove  cell.  The  zinc  is  placed  in  dilute  sulphuric  acid  in  the  outer 
vessel,  and  platinum  in  a  porous  vessel  which  contains  strong  nitric  acid  (depolariser). 
The  hydrogen  liberated  is  oxidised  to  water,  and  nitrogen  trioxide  fumes  are  given 
off  (the  great  disadvantage).  Voltage  1*9,  internal  resistance  may  be  as  low  as 
0-1  ohm. 

The  bichromate  op  Grenet  cell.  A  bottle-shaped  receptacle,  with  a 
neck  as  long  as  the  zinc  plate.  To  the  stopper  two  carbons  and  a  central  zinc  plate 
are  attached.  The  sensitising  fluid  is  dilute  sulphuric  acid,  as  in  the  other  batteries, 
whilst  for  depolarising  8  pc.  bichromate  of  potassium  is  added.  A  small  quantity 
of  mercurous  sulphate  in  the  solution  helps  to  maintain  the  amalgamation.  Both 
elements  dip  into  the  same  fluid.  From  this  the  zinc  can  be  withdrawn  when  the 
battery  is  not  in  use.  The  voltage  may  attain  to  over  2  volts,  but  soon  drops  and 
consequently  the  cell  is  only  of  use  for  short  periods.  The  internal  resistance  is 
low.  Disadvantage,  frequent  recharging  is  necessary,  and  creeping  of  the  fluid 
destroys  the  connection  of  the  carbon  with  its  terminals. 

Connections  op  leads.  No.  18  cotton-covered  wire.  The  ends 
must  be  bared  of  their  covering  and  cleaned  by  scraping  before  being 
attached   to   the   binding   screws   in   order   to   secure  good   contact. 

Never  omit  to  examine  the  wires  for  fractures,  especially  if  they 
are   wound   into   spirals. 

Keys.  These  are  inserted  into  the  circuit  wherever  it  is  desired 
to   control   the   passage   of  a   current. 


186 


ELECTRICAL    CONSIDERATIONS. 


The  Du  Bois  Eeymond  key  (Fig.  21,  A)  is  one  of  the  most  efficient, 
and   is   of   historical   interest. 

The  simple  switch  depicted  (Fig.  21,  B)  is  quite  as  efficient  and  is 
less  costlv. 


B 


WAKE     AND  BREAK 


Fig.  21.     A  Du  Bois  Reymoncl,  B  Simple  key. 

Whatever  the  key,  it  can  be  used  in  one  of  two  ways. 

1. — As  a  Make  and  Break  device  it  is  inserted  into  the  circuit  so 
that  when  closed  the   circuit   is   completed,  and  when   it   is  opened  the 

circuit  is  broken.  Always  use  it  in 
this  manner  when  in  circuit  with  a 
Leclanche   cell  (Fig.  22). 

2. — For  short  circuiting-  ("  short- 
ing"). In  this  case  the  circuit  is  never 
broken,  but  the  closure  of  the  kej-  is 
so  arranged  as  to  offer  a  path  of  less 
resistance   to   the  current. 

The  current  is  not   entirely  diverted 

from  the  "longer "  path,  but  practically 

so   little  passes    that    it    comes    to   the 

same    thing. 

Remember  that    in  a  divided   circuit   the   distribution  of   the  current 

is    proportioned    inversely    to    the    resistance   of    the    respective    paths. 

Les#    resistance  more   current ;    more   resistance   less   current. 

Commutator.  (Fig.  23.)  For  the  purpose  of  reversing  the  direction 
of   a   current.     Pohl's  instrument   is   the    best. 

The  rocker  consists  of  two  portions  joined  across  the  middle  line 
1>\     tn.     insulating   piece    R. 


"SHORTING" 
Fig.  22. 


COMMUTATOR. HAND    ELECTRODES. 


L87 


The  leading  in  binding  screws  are  attached  to  mercury  cups,  into 
which  the  middle  arms  of  the  rocker  are  placed.  For  the  purpose 
■of  reversing,  the  cross  wires  are  used,  and  the  leading  out  connections 
are  attached  to  the  binding  screws,  and  so  to  the  cups  on  one  side 
only.  When  the  arms  of  the  rocker  dip  into  these,  the  positive  pole 
remains  on  the  same  side ;  when  the  rocker  is  reversed  the  other 
leading   out   wire    becomes   positive. 


DEFLECTER 

FIG.  23.  Diagram  of  Pohl's  commutator.  The  rocker  R  is  shown  in  position 
for  reversal  of  the  current  in  the  left-hand  figure. 

To  use  the  Pohl  as  a  current  dejiecter  the  cross  wires  are  removed, 
the  connections  of  the  two  alternative  circuits  are  made  with  opposite 
pairs  of  cups,  and  the  rocker  changes  the  path  when  it  is  moved 
from   one   set   to   the   other. 


e7T^ 


Fig.  24.     Hand  electrodes. 


Hand  electrodes :— Two  stout  copper  wires  are  passed  through 
glass  tubes  for  insulation  and  rigidity.  The  ends  of  the  latter  are 
sealed  with  electric  cement ;  the  tubes  are  tied  together  and  their 
ends  are  covered  with  marine  glue,  the  free  ends  of  the  wire  are 
shaped  conveniently.  The  other  ends  are  soldered  to  flexible  wires 
which  terminate  in  tags  that  will  fit  either  screw-down  or  perforated 
terminals. 


188  ELECTRICAL    CONSIDERATIONS. 

9  Induction  machine.      Du   Bois   Reymond's  Induetopium. 

Principle  of  action  :— If  portions  of  the  wires  forming  two  separate 
circuits  be  placed  parallel  to  each  other,  as  in  the  case  of  the  planes 
of   the   two   spirals  or  coils  of  the  Inductorium  (Fig.  25),  the  one  wire, 


Fig.  25.  Induced  currents  are  only  shown  in  S.  The  solid  arrows  indicate 
the  direction  of  the  currents  at  closure  of  key  K.  Dotted  arrows  at  break. 
Similar  currents  occur  in   P  and  at  the  same  time. 

primary  (P),  being  connected  with  a  source  of  electricity  (battery),  the 
other,  the  secondary  (S),  being  simply  a  closed  circuit.  Whenever  the 
P  circuit  is  closed  (made)  or  is  opened  (broken)  currents  will  at  those 
moments   be   induced   in   the    S   circuit. 

The  make  induction  current  flows  in  the  S  circuit  in  a  direction 
opposite  to  that  in  the  P  circuit ;  whilst  the  break  induction  current 
flows  in   the   same   direction  as   the   original   battery  current. 

These   induction   currents   are   of  very   short   duration. 

Place  the  induction  machine  lengthways  in  front  of  you  on  the 
table   with   the   interrupter   turned   to   the   right. 

In  the  Du  Bois  Reymond  type  the  wires  are  wound  into  two 
separate  coils ;  the  P  coil  which  is  supported  by  a  wooden  upright 
attached  to  the  base  of  the  instrument  is  composed  of  relatively  thick 
wire,  whilst  the  S  coil  mounted  upon  a  sliding  foot  is  composed  of 
very  thin  wire,  in  this  case  invisible,  as  it  has  a  protective  covering 
<  )f   vulcanite. 

The  parallelism  of  the  wire  in  the  two  coils  is  maintained  so  long- 
as   the   axes   of  the   coils   coincide. 

The  successive  turns  of  wire  in  each  coil  are  also  practically 
parallel   to   each   other. 

The  P  coil  is  provided  with  a  core  of  soft  iron  wire  which 
magnetises  when  a  current  passes  in  the  surrounding  wire  an  electro- 
magnet  being   thus   formed. 

The  electrical  field  produced  by  the  coil  is  greatly  intensified  by 
this  core,   and   the   effect   on   the   S    coil    is    correspondingly    increased. 

The  nearer  the  S  coil  is  to  the  P  coil,  the  more  powerful  will 
be   the   induction   currents. 


INDUCTOKIU.M. 


189 


The  E.M.F.  of  the  currents  in  the  secondary  bears  a  direct  relationship  to  the 

E.M.F.  of  the  currents  in  the  primary.  Thus,  if  there  are  200  turns  in  the  P  and 
0,000  in  the  S,  the  E.M.F.  of  the  induction  currents  will  be  approximately  30  times 
that  of  the  inducing  currents,  independently  of  the  influence  exerted  by  the  iron  coi-e. 

Connect    up    the    secondary    circuit    of     the     Induetorium. 

(Fig.  26  S.)      It   is   well  to   do   this   in  all   cases   first. 

Fasten  a  key  to  the  table  close  to  the  left  -  hand  end  of  the 
machine  as  it  now  rests  on  the  table,  and  connect  the  binding  screws 
of  the  S  coil  with  those  of  the  key,  b}r  means  of  two  wires,  so 
that  when  the  key  is  closed  the  S  circuit  is  thereby  also  closed. 
This  is  the  short  circuiting  key  in  the  secondary  circuit. 

Now  attach  the  long  circuit  wires,  by  means  of  which  the 
connection  is  to  be  established,  with  the  seat  of  stimulation,  i.e., 
attach  the  hand  electrodes  by  their  metal  tags  to  the  binding  sciban  < 
which   already   hold  the   S   wires. 


Fig.  26.     Induetorium  of  du  Bois  Reymond. 


Connect  up  the  P  coil  fop  single  induction  currents*  (Fig.  26. ) 
Place  the  Leclanche  cell  upon  the  table  near  the  right  hand  end  of 
the   coil,    and   attach   a   key   to   the   table   close   to. 

Keep  the  key  open.  In  making  the  connections  always  begin  at 
the   batterv.    and   follow   the   direction   the   current   will  take. 


190  ELECTRICAL    CONSIDERATIONS. 

Connect  the  C  pole  of  the  cell  to  the  key  by  a  wire,  then  wire 
the  other  side  of  the  key  to  the  top  binding  screw  A  of  the  P  coil, 
wire    B   to   the   zinc   pole  of   the   cell. 

Withdraw  the  S  coil  to  20  cm  of  the  scale,  and  let  one  co-worker 
hold  the  electrodes  against  his  wetted  lip  whilst  the  other  makes 
the   trials. 

Make  and  break  the  P  circuit  with  the  key.  Do  this  smartly,  once 
or  twice  only,  and  after  each  trial  push  the  S  coil  half  a  cm 
towards   the    P   coil. 

Let  the  co-worker  indicate  when  he  feels  the  "shock,"  and  whether 
he  does   so   at   closure   or   at   opening. 

Note  the  position  of  the  coil  as  soon  as  the  minimal  break  shock 
is   felt ;    it   is   perceived  first. 

Proceed   with   further   trials   until   the   make   shock   is    also   felt. 
Read   off  the  position   of    the   S   coil. 
It   is   considerably   nearer   to   the    P   coil. 
The   break   shock     is   the   stronger  of  the   two. 

Continue  the  approximation  of  the  S  coil  by  short  steps  to  the 
P  coil,  the  shocks  will  be  stronger  each  time  until  finally  unbearable. 
The  strength  of  a  stimulus  can  therefore  be  varied  by  changing  the 
relative  position  of  the  S  coil.  It  may  approximately  be  assumed  to 
change  inversely  with  the  square  of  the  distance  between  the  two 
coils. 

Next  take  the  S  coil  out  of  the  slide  and  place  it  end  on,  and 
close  up  to,  the  P  coil.  Whilst  making  and  breaking  the  P  circuit 
turn  the  S  coil  so  that  its  axis  shall  be  ultimately  set  at  right 
angles   to   that   of   the    P   coil. 

The  shocks  will  rapidly  diminish  and  disappear  as  the  position  of 
the   S   coil   is   changed. 

Explanation: — When  the  battery  current  at  closure  of  the  circuit  is  rising-  in 
strength  in  the  primary,  an  opposing  induction  current  is  thereby  generated  in 
the  P  coil  itself,  which  retards  the  battery  current  from  attaining  its  full  strength 
as  soon  as  it  otherwise  would,  and  consequently  the  effect  upon  the  S  coil  is 
not  so  sudden  a  one. 

On  breaking  the  P  circuit  an  induction  current  is  likewise  generated  which 
has  the  same  direction  as  the  disappearing  battery  current,  and  consequently  it 
retards  change  of  the  electrical  condition  but  does  not  interfere  much  with  the 
suddenness  of  the  subsequent  drop  in  potential,  and  therefore  the  effect  upon  the 
S   coil  is  greater  than  at  closure. 

This  opening  extra  current  never  reaches  its  full  development,  as  it  originates 
at  the  moment  that  the  P  circuit  is  being  broken;  to  circulate  at  all  it  must 
needs  fly  across  the  air  gap  at  the  contact  E,  which  rapidly  increases  and  so 
quenches  the  flow. 


BREAK     EXTRA    CURRENTS. 


191 


It  is  well.  also.  t>>  bear  in  mind  that  contact-  are  by  no  moans  of  negligible 
duration,  for  they  vary  very  much  in  different  forms  of  instruments.  This  applies 
also    to   ordinary    keys. 

These  are  the  salient  features  in  the  action  of  the  Induetorium,  but  there  .ire 
others,  such  as  the  influence  of  the  electromagnet  of  the  interrupter,  dfcc,  which 
cannot    he   considered    here. 

Demonstration  of  the  break  extra  current  as  an  illustration  of 
one   of   the   induction   effects   in   the    P  coil  (Fig.  '27). 

Remove  the    S    coil   from    the  Induetorium   and    set  up  as   follows: 

First    set   up  the    Leclanehe   cell   in   circuit  with  the  hand  electrodes 
with   a    "shorting"   key   (1).     Hold    the   electrodes  against    your  tongue, 
and    open    and    close    the    key — nothing   will   be  felt.      The     E.M.F.    of 
the  current  is  not  sufficient  to  produce 
a  stimulus  through  the  resistance  offered 
by  the  skin. 

Now  add  to  the  battery  circuit  the 
P  coil  (slip  off  the  S  coil  and  place 
it  on  one  side)  with  shorting  key  (2), 
so  that  the  P  coil  may  be  at  will  in- 
or   excluded  from  the  circuit. 

Keep  key  (2)  closed,  and  repeat  the 
trial  as  before,  key  ^2)  excludes  the  coil 
from  the  circuit  and  the  result  is  the 
same  (effect  of  *' shorting").  Xext  open 
key  (2).  then  on  opening  key  (Da  shock 
is  felt  at  break.     This  shock   is   due  to 

the  induction  current  which  is  generated  in  the  coil  by  the  fall  of 
potential  in  the  battery  circuit.  The  fall  is  due  to  the  easier  path 
through  key  (1),  being  replaced  by  a  path  of  greater  resistance  through 
the  tongue,  and  consequently  a  feebler  current  flows  through  the  P 
circuit,  and  this  drop  generates  an  induction  current  in  the  circuit, 
and  the  latter  having  a  higher  E.M.F.,  is  able  to  pass  through  the 
tissues   and   to   act    as    a    stimulus. 

The  effect  is  largely  due  to  the  iron  cove  of  the  coil,  for  if  the  S  coil  be  sub- 
stituted the  effect  mil  not  be  so  great  in  spite  of  the  fact  that  this  coil  presents  in 
the  larger  number  of  its  turns  of  wire,  a  condition  favourable  to  the  production  of 
more  powerful  induction  effects. 

Interrupter  shocks.  Detach  the  wires  from  A  and  B  and  transfer 
them  to  the  binding  screws  C  and  D.  Adjust  the  top  contact  screw  E 
so  that  it  touches  the  spring  lightly  (Fig.  26b). 

On  closing  the  P  circuit  this  spring  oscillates,  automatically  opening 
and  closing  the  P  circuit,  and  a  succession  of  induction  currents  are 
generated  in  the  S  coil. 


Break  extra  current. 


192 


ELECTRICAL    CONSIDERATIONS. 


The  rate  of  their  occurrence  depends  upon  the  length  of  the  spring. 

Explanation  : — The  current  from  the  battery  flows  up  the  pillar  C, 
through  the  spring  up  through  the  top  contact  screw  to  the  P  coil,  and 
thence  round  the  electromagnet  F  and  back  by  the  base  of  D  to  the 
battery. 

When  the  current  flows  round  the  circuit,  F  is  magnetised  and  draws 
down  the  spring  I,  thus  breaking  the  top  contact. 

Upon  this  the  current  stops  flowing,  the  magnet  ceases  to  act,  the 
spring  is  released  and  again  makes  contact  with  E,  and  so  the  circuit 
is  re-established  and  the  cycle  begins  anew. 

As  the  break  shock  is  always  the  stronger  of  the  two,  it  follows  that 
if  these  shocks  are  passed  through  a  tissue  for  some  time  that  polarisa- 
tion effects  will  be  set  up.  Ordinarily  they  are  employed  for  a  short 
time  only,  and  this  effect  can  be  disregarded.  In  cases  in  which  this 
may  be  of  importance  the  next  arrangement  must  be  made  use  of. 


i. 


SINGLE 


INTERRUPTED      HELMH0LT2 


Fig.  28.     Connections  of  the  primary  circuit  (after  Waller). 


Helmholtz  wipe  for  the  equalisation  of  the  make  and  break  shocks 
(Fig.  28).  Leave  the  connections  as  for  interrupter  shocks  and  add  a 
wire  between  G  and  A.  Raise  the  top  contact  screw  E  clear  of  the 
spring,  and  turn  up  the  screw  on  pillar  D  until  it  touches  the  under 
side  of  the  spring  when  the  latter  is  held  depressed  against  the  electro- 
magnet.    On   closing   the  circuit   the   spring   oscillates. 

Explanation.— The  P  circuit  is  now  closed  at  all  stages  of  the  oscillations  of 
the  spring. 

When  the  current  first  enters  it  passes  round  the  long  path  through  the  P  coil 
and  F  is  magnetised.  The  spring  is  now  pulled  down  and  makes  a  shorting  contact 
;it  D.  The  current  leaves  the  longer  path  for  the  shorter  easier  one  C  to  D,  and 
is  practically  excluded  from  P  and  F.  The  .spring  is  released  arid  flies  back 
restoring  the  long  circuit,   and  the  cycle  begins  anew. 


HELMHOLTZ   WIRE.  193 

Both  extra  currents  in  the  P  circuit  now  gain  their  full  development,  and 
there  is  consequently  retardation* of  the  fall  of  potential  at  break. 

The  induction  currents  in  the  S  circuit  are  now  produced  by  fall  and  rise  of 
the  strength  of  the  currents  in  the  P  coil,  and  not  by  make  and  break.  The 
difference  in  the  change  is,  on  the  one  hand,  not  so  great,  and  consequently  the 
shocks  are  not  so  powerful;  but  on  the  other  hand,  owing  to  the  fact  that  the 
circuit  is  never  broken,  the  break  extra  current  gain!  its  full  development,  and 
there  is  consequently  a  retardation  of  the  fall  in  potential,  and  the  induced  shock 
in  the  S  coil  approximates  more  nearly  in  strength  to  that  at  closure. 

The  difference  is,  how  ever,  not  completely  removed  ;  the  opening  shock  remains 
a   little  stronger. 


DISSECTION    OF   THE   FROG. 


f-COC-IL 


note  its  tendinous  slip  over  PN.      T 


Fig.  29.  Dorsal  dissection  of  a  frog. 
E  Ear  ;  in  front  is  the  eye.  C  Callosity 
on  thumb  of  male.  Illiac  bone.  U  Urostyle. 
LH  Position  of  lymph  heart.  Nerves : — MCN 
Musculocutaneous  traversing  the  dorsal  lymph 
sac.  SN  Sciatic.  TN  Tibialis.  PN  Peroneal. 
SP  Sciatic  plexus.  Muscles:— COC- 1 L  Coc- 
ci geo-iliacus.  B  Biceps.  SM  Semi-membranosus. 
RIM  Rectus  interims  minor,  which  together 
with  rectus  interims  major  (see  Fig.  4)  is 
known  as  the  Gracilis.  G  Gastrocnemius, 
Tibialis.      TAc  Tendo  achillis.      SC  Sessamoid 


cartilage.     FT  Flexor  tendons  in  the  foot. 


CHAPTER    XXVIII. 

DISSECTION    OF    THE    FROG. 

Pithing-.  Stun  the  frog  by  a  sudden  blow  on  the  head  with  the 
stick  provided.  Pass  the  blunt  edge  of  the  knife  over  the  dorsal  surface 
of  the  head  until  the  groove  at  the  back  of  the  occiput  is  felt.  At  this 
point  and  in  the  middle  line  plunge  the  knife  transversely  into  the 
vertebral  canal,  so   as   to   sever  the   cord  (Fig.  29,  1). 

Make  the  incision  as  narrow  as  possible,  so  as  to  miss  the  vertebral 
arteries,    the   division   of  which  will   cause   much   bleeding. 

Pass  a  metal  seeker  upwards  into  the  cranial  cavity  and  turn  it 
round  freely,  so  as  to  disorganise   the  brain  completely.      Decerebratioit. 

To  complete  the  pithing,  pass  the  seeker  down  the  full  length  of  the 
canal,  so   as   to   destroy   the   spinal   cord. 

The  hind  limbs  will  twitch  violently  whilst  this  is  being  done,  but 
no  sensation  can  be  set  up,  as  the  brain  has  been  destroyed,  If  bleeding 
is  to  be  prevented,  plug  the  aperture  in  both  directions  with  pointed 
match  ends. 

Dorsal  dissection.  Lay  the  frog  on  the  loaded  frog  plate,  dorsal 
surface  uppermost  and  with  its  legs  extended. 

Find  the  longitudinal  groove  in  the  skin  in  the  middle  of  the 
thigh,  which  marks  the  position  of  the  biceps  muscle.  Under  this 
muscle   lies   the   sciatic   nerve. 

Pinch  up  the  skin  with  forceps,  make  an  incision  lengthways  up 
the  middle  of  the  thigh  to  the  illiac  bone  and  along  the  whole  of  its 
inner   edge.       Use  scissors  whenever  you  can. 

Next  prolong  the  incision  downwards  to  the  middle  of  the  foot. 
Pin  the  skin  aside  after  severing  the  few  attachments  at  thigh,  knee, 
and   heel. 

Note  the  dorsal  cutaneous  nerves  as  the  skin  of  the  back  is 
reflected.  The  skin  has  few  adhesions  to  the  subjacent  parts,  as 
extensive   lymph   sacs   intervene. 

Clear  the  narrow  biceps  carefully  from  its  neighbours  with  scissors, 
the  nerve  will  be  found  beneath  it,  crossed  from  without  inwards  by 
the   sciatic   artery. 


196  DISSECTION    OF    FROG. 

The  vein  almost  black  in  colour  accompanies  it  in  the  lower  part 
of  its  course,  then  leaves  it  above  to  run  outwards.  This  vein  after 
joining  a  transverse  branch  higher  up,  goes  to  form  the  renal-portal 
vein. 

Clear  the  nerve  carefully  from  its  surroundings,  lifting  it  with  the 
glass  seeker,  and  divide  all  restraining  tissue  around  it  with  scissors. 

Never  hold  the  nerve  itself  with  forceps,  and  do  not  -pinch  or 
pull  it  in  any  way.  Remember  that  this  is  a  living  structure 
and  very  easily  injured.  It  should  be  kept  moist  with  normal 
saline   and  must   on  no   account   be   allowed  to   dry. 

Two  branches  of  the  nerve  will  have  to  be  cut  in  the  thigh — one 
nearly  midway  externally,  and  one  higher  up  upon  its  mesial  aspect. 
Observe  that  muscles  twitch  when  this  is  done,  owing  to  the  mechanical 
stimulation  of  the  nerve  when  it  is  being  cut. 

In  severing  the  connective  tissue  where  the  nerve  enters  the  abdominal 
cavity,  keep  the  points  of  the  scissors  up  and  near  the  iliac  bone,  as  the 
nerve  lies  more  to  the  inner  side. 

Cut  through  the  coccigeo-iliacus  muscle  along  the  bone. 

Lift  the  end  of  the  urostyle,  clear  it  from  the  subjacent  "parts  (con- 
tents of  the  abdominal  cavity)  sever  it  from  the  spinal  column.  The 
sciatic  plexus  of  both  sides  are  now  in  view.  Cover  this  part  up  with 
a  flap  of  the  skin  and  proceed  to  clear  the  lower  end  of  the  nerve. 

When  the  skin  is  used  as  a  protective  covering  the  outer 
surface  must  not  be  placed  in  contact  with  muscle  or  nervey 
as   its   secretion   is  injurious  to   them. 

Near  the  knee  the  sciatic  divides  into  an  outer  peroneal  branch  which 
passes  under  a  tendinous  slip  from  the  gastrocnemius  muscle,  and  an 
inner  tibial  branch  which  turns  under  the  latter  muscle  to  be  distributed 
to  its  under  surface. 

Note  : — The  frog  is  to  be  employed  in  its  present  condition 
for  the  performance  of  those  experiments  which  can  be  carried 
out  without  recording.  For  recording  purywses  the  nerve-muscle 
preparation  requires  to  be  completed  as  follows  : — 

The  Nerve-muscle  preparation.  (Fig.  30.)  Where  the  Tendo- 
achillis  passes  round  the  heel  it  is  thickened  by  a  sessamoid  cartilage. 


NERVE    MUSCLE    PREPARATION. 


197 


Fig.  30.  G  Gastrocne- 
mius. H  S  hook  through 
tenclo  achillis.  F  Femur. 
S  Sciatic  nerve  with 
piece  of  spinal  column  C 
attached. 


Make  a  hole  through  the  latter  for  the  introduction  of  an   S  hook1 
by  passing   the   point  of   the   scissors    through  it  towards  the  subjacent 
bone.      Detach    the    tendon   and    sever   it   on    the 
tarsal   side. 

Raise  the  gastrocnemius  by  means  of  the  ten- 
don and  carefully  avoid  touching  the  muscle  with 
forceps. 

Clear  the  muscle  up  to  the  knee,  and  divide 
this  joint  transversely  with  scissors,  which  can 
be  done  without  danger  to  the  tibial  nerve  if  the 
muscle   be   turned   well   out   of   the   way. 

Next  clear  the  femur  of  all  muscles  excepting 
the  gastrocnemius,  and  sever  the  bone  near  the 
hip  joint. 

Divide  the  spinal  column  just  above  the  attach- 
ment of  the  nerves,  and  then  mesially  so  as  to 
leave   a  portion   attached   to   each  sciatic  plexus. 

Manipulate  the  nerve  by  means  of  this  piece  of  bone  and  sever  any 
restraining   tissue. 

Rapid  preparation  of  a  frog's  limb.  After  stunning  and  pithing  the 
frog,  hold  it  horizontally  by  the  hind  limbs  with  the  left  hand ;  the  fore  part  of  the 
body  will  hang  forming  a  sharp  angle  where  the  spine  meets  the  iliac  bones. 

Pass  one  point  of  a  pair  of  scissors  through  the  skin  under  the  middle  of  the 
spinal  column,  and  divide  the  back.  The  fore  end  will  then  hang  down,  held  by  the 
skin  on  each  side.  Divide  the  latter  the  whole  length  of  the  abdominal  cavity,  and 
then  cut  away  the  abdominal  contents,  taking  care  in  doing  so  not  to  injure  the  nerves. 

Grasp  the  skin  with  a  towel  at  its  spinal  end,  and  holding  the  spine  with  the 
ringers  of  the  other  hand  strip  the  skin  off  like  an  inverted  glove,  completely  denuding 
all  the  structures. 

Now  grasp  the  muscles  of  the  thigh  between  the  dried  ringer  and  thumb  of  the 
left  hand  so  as  to  put  the  dorsal  aspect  on  the  stretch,  and  scratch  through  the 
aponeurotic  tissue  until  the  nerve  is  exposed  completely  from  knee  to  sciatic 
opening,  and  complete  the  separation  of  the  nerve  and  muscle  as  before. 

Cut  away  all  the  muscles  from  the  femur,  divide  the  latter  at  its  upper  end,  raise 
the  limb  by  means  of  the  foot  so  as  to  lift  up  the  nerve,  cut  through  all  restraining 
tissue,  pass  one  blade  of  the  scissors  through  the  sciatic  opening,  divide  the  coccigeo 
iliacus  muscle  and  the  iliac  bone  at  both  ends,  lay  the  frog  on  its  back,  clear  the 
nerves  to  the  spine,  and  divide  the  latter  as  before. 

Clear  the  gastrocnemius,  insert  the  S  hook  and  remove  the  leg  as  before. 


1  Obtained  through  Messrs.  Reynolds  &  Branson,  Commercial  Street,  Leeds. 


CHAPTER    XXIX. 

EXPERIMENTS  ON  NERVE  AND  MUSCLE  WHICH 
CAN    BE    DONE    WITHOUT    RECORDING. 

Contraction  of  the  muscle  is  taken  as  the  index  of  the  excitation 
of  the  nerve. 

G-alvani's  experiment.  Raise  the  nerve  upon  the  metal 
seeker  and  with  the  latter  touch  one  of  the  pins  which  hold 
the  skin  down— a  contraction  of  muscles  will  occur.  Contraction 
ivith  metals. 

The  pin  and  seeker  form  a  galvanic  couple,  the  current  from 
which  stimulates  the  nerves  at  the  moment  of  contact  and  of 
separation.      Repeat  the  experiment  with  "electric  forceps." 

A  simple  form  of  the  latter  consists  of  a  copper  and  a  zinc 
wire  twisted  round  each  other,  the  free  ends  of  which  are 
separated  at  one  extremity  (Fig.   30). 


^^^@ 


Fig.  31.     Electric  forceps. 

Difference  of  make  and  break  induction  shocks.  Use 
single  induction  shocks,  slide  the  coil  to  30cm  and  find  the 
minimal  opening  shock  which  will  excite  the  nerve,  as  shown 
by   the   contraction    of   muscles. 

The    first   evidence   of   this    will    be    twitching   of    the    toes. 

Gradually  move  up  the  S  coil  until  the  induction  current 
at  closure  also  produces  an  effect,  and  note  the  distance  in 
each    case. 

Weaker  currents  are  required  than  were  necessary  in  the 
trials    upon    your   own    tongue.     The    nerve  is   more  excitable. 


RHEOSCOPIC    LIMB.  199 

Rheoscopic  limb.  Secondary  contraction.  The  nerve  must 
be  freshly  prepared.  Dissect  the  second  limb  of  the  frog  upon 
which  you  have  been  performing  the  previous  experiments,  and 
cut  the  nerve  near  the  vertebral  column. 

(a)  Let  the  nerve  fall  upon  its  own  muscle  in  such  a  manner 
as  to  touch  it  in  two  points,  one  of  which  is  near  the  middle 
(equator)  of  the  muscle  and  the  other  as  far  removed  from  it 
as  possible. 

A  contraction  will  follow  and  will  be  repeated  if  the  nerve 
is  very  sensitive  when  it  is  lifted  off  again.  Contraction  without 
metals. 

If  the  muscle  does  not  contract  make  an  artificial  cross  section 
and  let  the  second  point  of  contact  be  the  cross  section.  Defer 
this  second  part  until  (6)  and  (c)  have  been  concluded.  By  that 
time  the  excitability  of  the  end  of  the  nerve  may  have  disappeared 
and  it  will  be  necessary  to  cut  off  the  dead  portion. 

(b)  Place  the  rheoscopic  nerve  lengthways  upon  the  first 
muscle  and  stimulate  the  latter  with  interrupter  shocks.  The 
rheoscope  will    respond    to  every  contraction  of   the  first  limb. 

(c)  Lay  the  nerve  lengthways  upon  the  ventricle  of  the  frog's 
heart,  which  is  to  be  excised  for  the  purpose  and  must  be  beating 
vigorously. 

The  cause  in  (a)  is  that  the  rheoscopic  nerve  connects  two 
points  of  different  potential,  and  the  current  which  passes 
through  the  nerve  stimulates  it,  and,  secondarily,  its  own  muscle. 
In  (b)  and  (c)  the  rheoscopic  nerve  lies  along  the  path 
of  the  electrical  wave  which  courses  down  the  contracting 
muscle,  and  is  thereby  stimulated  and  its  muscle  contracts. 
The  rheoscopic  limb  acts  as  a  highly  sensitive  current  detector, 
hence    its    name. 

Transmission  of  nervous  impulses  takes  place  in  both   4 
directions  in   the  same  nerve.     Carefully    dissect    off    the 
muscles    of   the   anterior  surface    of   the   leg,  so   as    to   include 
the  distribution  of  the  peroneal  nerve. 


200 


MUSCLE    AND    NERVE. 


Detach  the  latter  as  far  up  to  its  junction  with  the  sciatic 
as  you  can,  lifting  the  nerve  by  means  of  the  muscle.  The 
tendinous  slip  from  the  gastrocnemius  will  have  to  be  severed. 

Lifting  the  nerve  by  means  of  the  muscle,  so  that  neither 
of  them  touch  the  frog  support,  stimulate  with  interrupter 
shocks  by  the  hand  electrodes,  and  increase  the  strength  of 
the    current    until    the    gastrocnemius    contracts. 

Great  care  must  be  taken  that  there  is  no  chance  of 
leakage  of  the  current  directly  to  the  muscle  or  its  nerve. 

It  is  often  difficult  to  convince  oneself  that  leakage  is 
not  the  cause.  In  case  the  doubt  should  exist,  apply  a 
ligature  to  the  peroneal  nerve  as  high  up  as  possible,  but 
clear  of  the  other  structures  of  the  thigh,  and  stimulate 
again.  If  the  ligature  has  been  drawn  sufficiently  tight, 
nerve  transmission  will  be  excluded,  and  leakage  will  offer  the 
only  explanation  of  the  phenomenon. 

Another  way  of  doing  this  experiment,  which  can  be  easily  tried  whilst 
both  sciatics  are  at  the  disposal  of  the  student  at  the  time  he  is  performing 
the  experiments  on  secondary  contraction,  is  as  follows  : — 

Lay  a  couple  of  cm  of  the  ends  of  the  two  sciatics  parallel  and  in 
contact  with  each  other  upon  a  small  block  of  paraffin,  the  latter  being 

of  such  dimensions  that  it 
will  leave  between  itself  and 
the  muscles  on  each  side  a 
space  of  36m. 

Stimulate  the  nerve  near 
one  muscle.  Lift  the  muscle 
A  from  the  plate  (to  prevent 
leakage  of  current  along  the 
plate  to  the  opposite  muscle) 
and  stimulate  the  nerve  at  S 
as  far  from  the  paraffin  block 
as  possible.  If  the  nerves 
arc  in  a  normal  condition  induction  shocks  of  considerable  strength  will 
have  to  be  applied  before  any  effect  is  produced,  and  the  limit  of 
these  must  not  overstep  the  strength  at  which  leakage  occurs. 


Pig.  32. 

P  paraffin  block,  W  waxed  paper,  E  electrodes. 


EXCITATION    OF    NERVES.  201 

Strength  of  interrupter  shocks.  Slide  the  S  coil  to 
35 cm  and  note  its  position  when  the  first  manifestations  of 
stimulation    are    apparent. 

The  distance  of  the  S  coil  will  be  greater  than  with  the 
single  shocks.  This  is  chiefly  due  to  the  quicker  and  more 
regular  make  and  break  of  the  P  circuit,  especially  when 
using  a  Leclanche  cell,  which  does  not,  under  these  circumstances, 
remain  close  circuited  for  so  long  a  time,  and  in  consequence 
the  potential  of  the  battery  does  not  drop  to  any  appreciable 
extent. 

It  is  also  due  to  the  rapid  repetition  of  shocks  which,  if 
applied    singly,   are   ineffective. 

Effect  of  stimulating"  different  nerves.  Stimulate  the 
roots  of  the  sciatic  separately,  near  the  spine,  with  interrupter 
shocks. 

Also  the  peroneal  and  the  tibial  branches.  Next  the  trunk 
of  the  sciatic  with  various  strengths  of  current,  and  note  the 
difference  produced  upon  the  class  of  muscle  called  into  play, 
i.e.,  extensor  and   flexors. 

Excitation  of  a  muscle  by  various  stimuli  other  than 
a  nervous  one. 

(a)  Mechanical.  Strike  the  gastrocnemius  smartly  with  the 
handle  of  a  scalpel,  the  muscle  will  contract.  The  blow  must 
be    a   sharp  one. 

(b)  Chemical.  Dissect  out  the  sartorius  muscle,  hold  it  with 
forceps  over  a  glass  rod  which  has  been  dipped  in  ammonia 
so  that  the  vapour  may  play  on  the  muscle.  The  muscle 
will  curl  up  in  a  continued  contraction  (contracture).  Ammonia 
kills    nerve   without    stimulating    it. 

Excitation  of  nerve  by  various  forms  of  stimuli. 

(a)  Mechanical.  Whenever  a  nerve  is  cut,  a  twitch  of  its 
muscle   is   evidence   of   its   stimulation.     In    this    manner   when 


202 


MUSCLE    AND    NERVE. 


the  nerve  is  suddenly  pulled  or  struck,  it  is  thrown  into 
action.  Try  these  forms  of  stimuli  last,  upon  a  nerve  which 
has  been  in  use  for  some  other  experiment. 

(b)  Thermal  stimuli.  Heat  a  stout  copper  wire  in  a  flame, 
and    apply  it    suddenly  when    very  hot    to    the   nerve. 

(c)  Chemical  stimuli.  Apply  a  drop  of  saturated  chloride 
of  sodium  to  either  the  end  or  to  some  other  part  of  a 
nerve,  its  muscle  will  commence  twitching  and  will  soon  pass 
into  a  continued  contraction  (salt  tetanus).  The  contractions 
are,  however,  not   completely  fused. 

Changes  in  the  excitability  of  a  nepve  when  dying.  Lay 
beneath  the  whole  length  of  the  sciatic  nerve  a  strip  of  waxed 
paper,  and  keep  it  moist  with  normal  saline. 

Carefully  raise  the  nerve  with  the  glass  seeker  and  explore 
it  from  end  to  end  with  minimal  single  induction  shocks,  the 
effect  of  which  have  been  tested  first  in  the  middle  of  the  nerve, 
and  note  if  there  be  a  difference  of  excitability  at  any  point. 
There  usually  is  at  one  or  two  points — find  them. 

Be  guided  in  your  estimation  of  this  by  change  in  the  mus- 
cular effect  evoked,  such  as  increase,  diminution,  or  absence  of 
contraction. 

Next  cut  the  nerve  at  its  spinal  origin,  and  compare  the 
excitability  at  the  cut  end  with  that  at  a  point  near  the  muscle. 
Repeat  this  from  time  to  time.  The  cut  end  will  presently 
exhibit  a  greater  excitability,  which  will  fail  later  until  it  is 
completely  lost. 

A  dying  nerve  at  first  rises  and  then  falls  in  excitability, 
finally  losing  it  altogether.  Remember  that  a  nerve  which  is 
drying  becomes  more  irritable   for   this  reason. 

Relative  excitability  of  muscle  and  of  nerve.  Find  the 
minimal   shock  which  will  evoke  a   muscle   twitch   through  the 


EXCITABILITY    OF    MUSCLE    AND    NERVE.  203 

nerve,  and  then  apply  the  same  stimulus  to  the  gastrocnemius 
directly.      It   will    be    ineffectual. 

Approach  the  S  coil  until  the  stimulus  is  strong  enough 
and    note    the    difference    in    strength    required. 

From  this  experiment  alone  it  is  not  permissible  to  conclude 
that  muscle  itself  is  directly  stimulable,  the  participation  of 
nerve  endings  in  muscle  not  being  excluded  from   the   process. 

See   the   experiment  with    Curara   later. 

Induction  shocks  have  great  power  of  overcoming" 
resistance.  Place  a  nerve-muscle  preparation  in  a  muscle 
chamber  at  one  end  of  the  room.  Lay  the  nerve  upon  the 
platinum  electrodes  and  connect  one  of  its  poles  by  means 
of  a  long  wire  to  one  of  the  terminals  of  the  S  coil  of  the 
inductorium.  The  latter  is  to  be  placed  on  a  table  at  the 
furthest  distance  from  the  muscle-chamber  which  the  room 
will  allow,  and  is  to  be  insulated  on  inverted  porcelain  basins. 

Let  your  co-worker  make  and  break  the  P  circuit,  or  use- 
interrupter  shocks — the  muscle  does  not  contract — the  insula- 
tion  is  sufficient  to  prevent  the  passage  of  the  current. 

If  he  now  touches  the  unconnected  terminal  of  the  S  coil 
with  his  finger  the  muscle  will  contract.  His  body  by 
bridging  the  gap  in  the  S  circuit  between  the  floor  and  the 
binding  screw  allows  enough  current  to  pass  to  stimulate 
the    nerve. 

This  so-called  unipolar  stimulation  is  therefore  due  to 
defective  insulation  and  is  the  reason  for  which  the  S 
circuit  must  always  be  provided  with  a  shorting  key,  since 
the  insertion  in  this  circuit  of  an  open  break  key  is  not 
enough  to  prevent  induction  currents  from  passing  to  the 
preparation,  and  especially  so  when  strong  currents  are  in  use. 


V  .. 


6 


CHAPTER    XXX. 

ARRANGEMENTS    FOR    RECORDING. 

General  arrangement  of  the  work  table.  The  typical 
method  of  distributing  the  apparatus  upon  the  table  is  shown  in  Fig.  33. 
Battery  to  the  right,  inductorium  in  the  centre  near  the  edge  of  the 
table.  P  the  key  in  the  P  circuit,  S  that  in  the  S  circuit.  The 
recording  cylinder  and  stand  are  conveniently  placed  to  the  left  so 
that  they  may  be  easily  accessible. 


Fit;.  33.      Work  table. 


On  the  table  shelf  heneath  to  the  left  of  F  the  leaded  frog  plate 
to  its  right  the  porcelain  bowl  covered  with  a  plate  in  which  the  frog 
is  placed  until  wanted.  W  is  the  drawer  for  wires,  upon  its  edge  the 
keys  are  supported  when  not  in  use.  The  inductorium  is  placed  to  the 
right  of  W  when  not  in  use.  N  normal  saline,  and  to  the  right  is 
suspended  the  stick  for  killing  frogs  or  for  coiling  wires. 
The    Recording-   Cylinder    (Fig.  34). 

1.    The  drum  (diameter  0  inches)  can  be  adjusted  for  height  on  its 
axle,   and    is  secured    in  position  by  tightening  a  thumb-screw.     It    can 


RECORDING    CYLINDER. 


205 


be  slipped  off  the  axle  for  covering  with  paper  and  smoking  the  surface 
of  the  latter.  The  axle  runs  in  double  ball  bearings  at  its  lower  end. 
•2.  The  driving  pulley  D,  with  various  speeds  (grooves  for  the 
driving  cord),  is  carried  on  an  axle  which  runs  on  ball  bearings  C,  and 
transmit-,  its  motion  to  the  driving  disc  on  the  drum  axle  by  means  of 


FIG.  34.      Cylinder,  general  stand  and  driving  cord. 

a  roller  E.  The  bearings  which  support  this  axle  are  pivoted  to  an 
upright  on  the  drum  stand,  and  an  arm  B  actuates  the  rocker  in  such, 
a  fashion  as  to  start  and  stop  the  drum  without  arresting  the  move- 
ment of  the  driving  pulley. 


206 


RECORDING. 


3.  Automatic  contact.  A  pin  G,  which  projects  from  the  edge  of 
the  drum  disc,  strikes  against  a  Avire  H  once  in  the  revolution  of  the 
drum.  This  wire  is  so  bent  as  to  allow  the  pin  to  pass  in  either 
direction.  In  setting  the  contact  it  must  be  made  as  short  as  possible, 
so  that  the  make  and  break  shocks  which  are  produced  may  be  fused . 
into  one  stimulus. 

One  contact  in  a  revolution  is  sufficient  for  the  purposes  of  these 
exercises,  but  another  arrangement  can  be  substituted  which  allows  of 
single  or  successive  stimuli  at  different  intervals. 

Recording"  arms  and  points.  Thin  wooden  rods  2mm  thick 
armed  at  one  end  with  an  aluminium  point  A  of  the  accompanying 
pattern,  the  actual  end  of  which  consists  of  a  glass  thread  1 cm  long. 
Its  end  is  glazed  to  perfect  smoothness  in  a  small  flame,  and  is  then 
attached  by  cement. 


22' 

H 


B 


|Q9 


Fig.  35.      H  Hinge.      A  Writing  point.      B  Silk  double  loop. 

Two  pieces  of  the  wooden  rod,  one  22 cm  and  the  other  8 cm  long 
are  inserted  into  the  outer  holes  of  the  hinge  piece  of  the  muscle- 
chamber  and  held  together  with   thread  as  in  the  figure. 

This  arrangement  is  light,  strong,  and  possesses  great  horizontal  and 
vertical  rigidity,  the  necessary  play  being  provided  by  the  aluminium 
point. 

Attachment  of  the  muscle  to  the  recorder.  Tie  a  piece  of  silk  thread 
(Fig.  35,  B)  (fine  plaited  roach  line  is  best)  into  two  loops  each  2*5 cm 
long,  the  whole  not  to  exceed  6  cm  in  length 

Draw  the  knots  tight  to  prevent  stretching. 

Fasten  one  end  to  the  lever  by  placing  one  loop  round  the  latter 
and  passing  the  other  through  it  and  draw  it  tight. 

Let  the  thread  rest  at  such  a  distance  from  the  hinge  that  the 
movement  of  the  point  shall  magnify  the  movement  of  the  muscle  four 
or  five  times. 

Pass  the  other  end  through  the  hole  in  the  floor  of  the  muscle- 
chamber  and  slip  it  over  the  hook  on  the  tendon. 

Covering-  the  drum.  Take  a  sheet  of  the  ready  cut  glazed  paper 
from  the  tin  on  the  shelf  of  the  varnishing  table  (Fig.  3(3)  and   lay  it, 


SMOKING    CHAMBER. 


207 


glazed  surface  down,  on  the  table  ;  wrap  it  evenly  round  the  cylinder, 
and  fasten  it  on  tightly  by  means  of  the  gummed  edge  after  the  manner 
of  a  newspaper  wrapper. 

Smoke  the  surface  of  the  paper.     A  special  chamber  (Fig.  36),  which 
prevents  access  of  smoke  to 
the  room,  is  provided  in  the 
wall  of  the  laboratory. 

Take  the  spindle  A  from 
the  chamber,  mount  the 
drum  upon  it,  clamping 
screw  to  the  right,  and 
place   in   the   chamber. 

Uncover  and  light  the 
wick  (6  inch)  of  the  lamp 
beneath  the  smoking  cham- 
ber, adjust  the  flame  so  as 
to  cause  a  uniform  sheet 
of  smoke  to  play  upon  the 
paper.  Close  the  door  C 
of  the  chamber,  revolve 
the  cylinder  at  about  once 
a  second.  Inspect  through 
the  door  from  time  to  time, 
and  extinguish  the  flame 
when  the  paper  is  uniformly 
covered  with  a  deep  brown 
covering  of  soot.  The  cover- 
ing must  not  be  thick  on 
account  of  the  resistance 
which  it  offers  to  the  writing 
point.  Therefore  stop  short 
of  complete  blacking. 

Injury  to  the  blacking 
on  the  cylinder  when  with- 
drawingit  from  the  chamber 
is  prevented  by  the  guides. 

The  natural  draught  may  be  so  great  that  it  may  be  necessary  to  keep  the  aperture 
at  D  open.  In  case  of  a  down  draught  there  is  a  ring  burner  in  D,  by  means  of  which 
the  upward  current  can  be  ensured. 

The  chronogram.  Though  a  time  tracing  may  be  inscribed  by 
means  of  a  point  attached  to  a  tuning  fork  or  other  oscillator,  writing 
directly  upon  the  recording   surface,   an   electrically  driven   style  is  the 


Fig.  36.      Smoking  chamber. 


•20$ 


RECORDING. 


TB 


Fig.  3S.  F  96  a  sec.  tuning  fork 
S  10  a  sec.  spring,  B,  B  their  driving- 
batteries.  TK  keys  carried  by  F  and  S 
bridging  the  table  circuits  (mercury 
cups)  and  dry  contact  in  the  clock. 
TB  main  table  circuit  battery  joined  in 
parallel  as  required,  SG  electric  style, 
TP  plugs  numbered  to  correspond 
with  the  tables  and  by  means  of 
which  the  style  is  placed  in  circuit 
with   TK  of   either  clock,    F  or  S. 


1T<;.  37. 


Time  distribution  boa 

the  laboratory. 


up  in 


CHRONOGRAPH.  209 

most   convenient.      The  apparatus  consists  of  (1)  the  time  giver  and  (2) 
the  repeater,   or  style,   which  writes  upon  the  recording  surface. 

For  the  purpose  of  supplying  a  number  of  workers  simultaneously 
the   following   device    is   adopted    in   this   laboratory  : — 

A  time  distribution  board  is  placed  against  the  wall,  which  consists 
of  a  clock,  the  pendulum  of  which  beats  seconds.  In  the  case  below 
are  a  tuning  fork  which  oscillates  9(3  times  and  a  spring  oscillating 
10    times    a    second. 

Both  the  latter  are  kept  in  action  by  independent  Leclanche 
batteries  (one  quart  cell  to  each,  two  are  available  in  case  of  need). 

The  clock,  fork,  and  spring  control  a  main  table  circuit,  which 
consists  of  a  battery  of  ten  Leclanche  cells,  the  leads  of  which  branch 
to  the  tables,  where  they  terminate  in  fixed  binding  screws. 

A  worker  at  any  of  the  tables  can,  after  connecting  an  electric 
style  to  the  binding  screws  of  his  table,  select  any  of  the  above  time 
fractions  by  placing  the  plug  which  corresponds  to  his  table  number 
into  the  brass  bar  of  the  time  board. 

The  accompanying  diagram  explains  the  method. 

Electric  style.  Chronograph.  This  consists  of  an  electro-magnet, 
to  the  armature  of  which  a  writing  point  is  attached.  The  latter  is 
drawn  away  from  the  magnet  by  means  of  a  spring,  the  tension  of 
which  is  overcome  whenever  the  current  passes  through  the  coil.  The 
core  of  the  latter  is  made  short  and  of  electrolysed  iron,  so  that  it 
shall  have  no  magnetic  memory,  i.e.,  will  demagnetise  the  moment  the 
current  ceases  to  flow  through  it. 

The  form  used  is  Smith's  style,  which  possesses  the  great  advantage 
of  having  a  low  resistance,  its  coils  being  wound  in  parallel  and  in 
having  its  armature  placed  at  the  end  of  a  comparatively  long  arm. 
The  result  is  that  it  possesses  a  quick  response  and  the  "lost  time'"  is 
very  short,  even  though  comparatively  small  electrical  power  be  emplo3Ted. 

Muscle  chamber.  (Fig.  34.)  A  wooden  floor,  with  a  glass  cover, 
having  a  screw  nut  underneath  for  attachment  to  a  bracket  P  on  the 
pillar  of  the  general  stand. 

A  brass  upright  pierces  the  floor  and  carries  within  the  chamber  a 
clamp  for  holding  the  femur  of  a  muscle  preparation,  and  beneath  a 
hinge-piece  to  which  the  writing  arm  is  fixed.  These  are  adjustable 
vertically  and  laterally.  Care  should  always  be  taken  to  set  the  arm 
horizontally  at  the  commencement  of  a  record. 

Inside  the  muscle  chamber  are  also  platinum  and  non-polarisable 
electrodes  upon  a  horizontal  bar,  not  shown  in  the  figure. 

General  stand.  (Fig.  34.)  A  heavy  foot  supports  an  upright,  to 
which  a  stout  pillar  of  steel  N  is  pivoted  between  centres. 


210 


RECORDING. 


This  pillar  can  be  turned  by  means  of  an  arm  O,  which  moves 
against  a  ridge  with  a  stop  at  its  end  above  M.  When  setting  writing 
points  always  bring  the  arm  up  to  this  stop.  The  points  can  then  by 
moving  O  be  removed  from  the  writing  surface,  and  can  be  returned 
to  it  without  losing  their  setting. 

The  pillar  carries  a  bracket  P  for  the  attachment  of  muscle  chamber 
or  frog-heart  recorder,  for  which  purpose  the  latter  carry  clamping 
screws.  An  extension  piece  is  sometimes  useful,  which  fixes  to  the 
bracket  and  the  above  for  more  convenient  adjustment  of  points  when 
very  short  writing  arms  are  employed. 

The  side  bar  Q,  a  rod  bent  D  shape  is  also  carried  by  the  pillar 
for  use  with  time  marker,  &c.     (See  Figs.  34,  40.) 

Fixing"  Tracing's.      The   soot   is   fixed   to   the   paper   by   means   of 

resin  (15p-c)  dissolved  in  methylated  spirit. 

Free  the  paper  from  the  cylinder,  so  as  not  to  touch  the  smoked  side,  as 

follows : Remove  the  cylinder  from  its  axle,  hold  it  with  the  left  hand 

horizontally  and  rest  the  clamping 
boss  upon  the  edge  of  the  table, 
place  the  thumb  of  the  left  hand 
on  the  overlap  of  the  paper,  and 
pass  the  point  of  a  sharp  knife 
under  it  along  the  line  of  junction, 
keeping  the  cutting  edge  turned 
away  from  the  cylinder. 

The  detached  end  of  the  paper 
should  fall  clear  of  cylinder  and 
table,  still  held  by  the  left  thumb. 
Raise  the  cylinder  and  paper  over 
the  table  and  lay  the  paper  down, 
smoked  surface  uppermost. 

Next  fill  in  such  written  details 
as  may  be  desirable  concerning  the 
manner  of  carrying  out  the  experi- 
ment, date,  name,  &c. ,  writing  with  a 
smooth  point  through  the  blacking. 
Take  your  tracing  to  the  var- 
nishing table,  place  a  pool  of  varnish 
one  inch  deep  in  the  trough.  Hold 
the   paper   by   both   ends,  smoked 

surface  uppermost,  bring  the  hands 
Fig.  39.    Varnishing  table.     P  paper.  L  i  '  ° 

v  nuntth.    F  frame.    B  hanging  rods.  together  so  as  to  form  the  paper 


FIXING    TRACINGS.  211 

into  a  loop,  and  dip  its  centre  into  the  varnish.  Guide  it  from 
end  to  end  through  the  latter  until  the  whole  is  saturated,  and  drain 
off  the  flowing  varnish  by  holding  the  paper  by  one  end  and  touch 
the  blank  side  of  the  other  against  the  edge  of  the  trough.  Transfer 
to  the  drying  frame,  and  fasten  with  pins.  Hang  the  frame  bj^  its  hooks 
on  the  rods  of  the  table.  The  tracing  will  be  dry  in  about  15  minutes. 
When  dry,  cut  out  the  portion  which  is  to  be  preserved  and  paste  it  into 
your  book  on  the  blank  page  provided  for  it. 


7  CHAPTER   XXXI. 

EXPERIMENTS  ON  MUSCLE  (WITH  RECORDS). 

Extensibility  and  elasticity  of  muscle. 

Required:—  General  stand  with  muscle  chamber,  drum,  ten  10 g 
weights  (split  lead),  nerveless  muscle  preparation. 

Set  up  as  follows : — Attach  the  muscle  by  its  femur  to 
the  clamp,  and  fasten  the  tendon  by  a  silk  thread  to  the 
recording  arm,  clear  of  the  aperture  in  the  chamber  floor. 
Place  moist  blotting  paper  on  the  inner  side  of  the  glass 
cover,    and    put    the    latter    into    position. 

Adjust  the  writing  arm  horizontal ty  and  set  the  pillar 
arm  against  the  stop ;  apply  the  writing  point  to  the  drum ; 
this  adjustment  should  always  be  made  near  the  paper 
overlap. 

Suspend  the  hook,  to  carry  the  weights,  from  the  writing 
arm    close    to    the    muscle    attachment. 

Turn  the  cylinder  by  hand  for  2  or  3cm  so  as  to  write 
a    short    abscissa.     Return    to    the    starting   point. 

Next  add  successive  weights ;  after  each  let  the  muscle 
attain  its  full  extension,  and  then  move  the  recording  surface 
7    or    8mm   onwards   before   adding   the   next   weight. 

The   muscle   will    extend    less   with   each   additional    weight. 

When  all  the  weights  have  been  applied,  proceed  to  remove 
them  after  the  same  fashion. 

The  muscle  will  shorten  by  elastic  reaction,  which  towards 
the    end    is    very    slow    in   effecting    complete    restoration. 

Extensibility  of  muscle  increases  during*  contraction. 

Required: — General  stand  with  muscle  chamber,  drum,  inductorium, 
Leclanchc  cell,  2  keys,  7  ordinary  and  2  thin  wires,  nerveless  muscle 
preparation,  ten  \()«  weights. 


SINGLE    MUSCULAR    CONTRACTION.  213 

Set  up  as  follows  : — Arrange  for  single  shocks,  connect  the 
S  circuit  to  the  outer  terminals  of  the  muscle  chamber  and 
substitute  the  fine  wires  for  the  electrodes.  Clamp  the  muscle 
into  position  and  attach  it  to  the  writing  arm,  and  make 
one  of  the  thin  wires  fast  to  the  femur,  and  the  other  to 
the   tendon;    the    second   wire  must  not   constrain  the  muscle. 

Choose  single  induction  shocks  to  produce  strong  contractions. 

Take  only  the  break  shocks,  e.g.,  close  the  S  key  each 
time  before  closing  the  P  circuit,  and  open  the  former  again 
before  the  P  circuit  is  opened.  These  evolutions  should  be 
performed  in  rapid  succession,  so  as  not  to  keep  the  P 
circuit    closed    for    long    (to    ensure    equal    stimuli). 

Apply  a  weight,  let  the  muscle  extend  fully,  turn  the 
drum  by  hand  some  7 mm,  stimulate  the  muscle ;  it  will  draw 
an  upstroke  and  will  on  relaxation  be  extended  below  the 
line  from  which  it  started.  Turn  the  drum  onwards  for 
the  same  distance  as  before,  add  another  weight  and  repeat 
the  process  until  all  the  weights  have  been  added. 

Examine  the  tracing  and  compare  the  passive  extension 
due  to  the  application  of  the  weight  with  that  which 
accompanies    relaxation    after    each    contraction. 

Single    muscular    (isotonic)    contraction    and    latent   8 
period,  with    direct   excitation    of    the   muscle. 

Required: — Muscle  chamber  on  stand,  inductorium,  electric  style, 
2  Leclanche  cells,  3  keys,  10  wires  (two  of  them  fine).  Drum  to  fastest 
speed.     Muscle  load  10s- 

1.  Set  up  for  single  induction  shocks  and  include  the  drum 
contact  in  the  P  circuit.  See  that  the  contact  is  very  slight, 
so  that  the  make  and  break  shocks  may  be  fused  into  a 
single  stimulus.  Keep  S  key  closed  until  the  time  of  stimula- 
tion. Detach  the  platinum  electrodes  from  their  binding  screws 
in  the  muscle  chamber  and  substitute  the  fine  wires  for  them. 


214  MUSCLE. 

2.  Attach  the  electric  style  to  the  side  bar  of  the  stand 
and  wire  it  to  the  table  binding  screws.  Include  a  key 
in  this  circuit.  Put  the  plug  corresponding  to  your  table 
number  into  the  96th  seconds  of  the  time-board,  close  the 
key,  and  ascertain  that  it  works  properly.  The  time  record 
may  be  written  directly  beneath  and  simultaneously  with  the 
muscle  curve  if  great  exactitude  is  required.  It  is  not 
necessary  to  do  so  in  the  present  case,  as  the  drum  will 
revolve  with  a  constant  speed  during  the  performance  of  the 
experiment,  which  only  takes  a  short  time  to  carry  out.  It 
may  be  inscribed  immediately  before  or  after  the  muscle 
tracing. 

3.  The  muscle  preparation  is  made  as  previously  directed, 
but  the  nerve  is  cut  off  close  to  the  muscle  (nerveless  muscle). 

After  fixing  to  the  clamp,  connecting  the  tendon  to  the 
writing  point,  and  applying  the  load,  hook  one  thin  wire 
round  the  tendon  and  one  round  the  femur.  The  former 
must  not  impede  the  movement  of  the  muscle  nor  draw  it 
to    one   side.     The    wire    should    be    wound   into   a  spiral. 

4.  Mark  the  paper  overlap  at  the  top  by  sweeping  off  some 
of  the  blacking  with  your  finger.  This  is  to  act  as  a  guide 
for  the  application  of  the  writing  points  when  the  drum  is 
revolving. 

Set  the  latter  so  that  the  drum  contact  shall  occur  when 
the   writing   points  are  clear  of    the  overlap  by   10 cm- 

5.  Adjust  the  writing  points  : — Draw  the  stand  clear  of  the 
drum,  push  the  handle  which  rotates  the  pillar  up  to  the 
stop,  see  that  the  muscle  chamber  is  firmly  fixed.  Arrange 
the  points  of  muscle  and  time  recorders  to  write  in  the  same 
plane  and  as  near  to  each  other  as  possible,  draw  the  drum 
up  to  the  stand  until   the  recorders  are  in  equal   contact  with 


LATENT    PERIOD.  215 

it.  They  must  write  on  the  same  vertical  line.  .Make  these 
adjustments  on  the  paper  close  to  the  overlap  so  as  not  to 
encroach  on  valuable  space.  Remove  the  points  from  contact 
with  the  paper  by  turning  the  pillar  arm  :  the  points  will 
fall  back  into  position  when  the  arm  is  again  brought  up  to 
the    stop. 

6.  Take  a  record.  Start  the  style  by  closing  its  key  ;  start 
the  drum,  let  it  run  round  three  times  so  that  it  may  settle 
to  its  speed ;  open  the  S  key,  note  if  the  muscle  gives  a  good 
contraction,  and  if  its  does,  apply  the  points  to  the  paper 
the  moment  that  the  guide  mark  comes  into  view,  and  lift 
them  off  again  on  its  next  appearance.  The  record  thus  lasts 
for    one    revolution    only. 

Examine  the  curve: — The  muscle,  if  unfatigued,  will  record 
an  even  sweep  upwards  to  a  summit  and  a  symmetrical  fall. 
The  lever  will  not  return  at  once  to  the  line  from  which  it 
originally  started,  but  after  the  first  symmetrical  fall  will 
exhibit  a  slower  contraction  remainder.  This  is  not  always 
a  smooth  descent,  but  frequently  consists  of  several  oscillations, 
the  first  of  which  falls  below  the  abscissa  to  rise  above  it 
again.  This  may  be  repeated  several  times  in  very  vigorous 
muscles.  They  disappear  with  fatigue.  Should  the  upstroke 
of  the  curve  show  an  indentation  on  its  ascent  or  a  double 
crest,  the  drum  contact  is  at  fault  and  should  be  reset, 
because  the  opening  shock  has  been  delayed  through  the 
length  of  the  contact,  and  two  separate  stimulations  are  being 
produced. 

7.  Measure  the  latent  period.  Turn  the  style  aside  to 
prevent  it  writing,  and  replace  the  muscle  lever  in  contact 
with  the  paper  at  the  beginning  of  the  tracing.  Carry  an 
abscissa   beneath    the   whole  length    of    the   former.      Lift   the 


216  MUSCLE. 

point  off  again,  bring  the  paper  to  the  starting  point  and 
re-apply.  Open  the  S  key,  close  the  others,  and  slowly  turn 
the  drum  by  hand  until  contact  occurs ;  the  muscle  lever 
writes  an  upstroke.  This  is  the  moment  in  the  tracing  at 
which  the  stimulus  was  given.  Turn  off  the  muscle  recorder, 
close  S  key.  Next  mark  the  exact  position  at  which  the 
recorder  began  to  rise,  and  measure  the  interval  by  com- 
paring  it   with    the   time    tracing. 

The  latter  consists  of  oscillations  in  which  each  down 
stroke  of  the  style  is  a  sharply  denned  descent.  The  in- 
tervals between  these  are  96th  parts  of  a  second ;  estimate 
fractions   by   the   eye. 

Tetanus.     Summation  of  muscular  contractions. 

Required : — Drum,  muscle  chamber  on  stand,  inductorium,  variable 
spring,  2  Leclanche  cells,  8  wires.  Drum  at  a  middle  speedy  Nerve 
muscle  preparation,  with  indirect  stimulation. 

Set  up : — S  circuit  as  usual,  P  circuit  with  the  variable 
spring  in  the  position  of  the  usual  break  key ;  it  is  to  act 
as    a    mercury   key. 

1.  First  record  two  or  three  muscular  contractions  clear  of 
each  other,  but  on  the  same  line,  by  making  and  breaking 
the  P  circuit.  Do  this  by  pressing  the  needle  of  the  variable 
spring  in  and  out  of  the  mercury  cup. 

Next  throw  several  stimuli  in  rapid  succession  into  the 
nerve,  moving  the  spring  by  hand  as  before ;  these  will 
succeed  each  other  irregularly,  but  will  be  sufficiently  rapid 
to  cause  the  contractions  to  follow  each  other,  so  that  the 
muscle  has  not  time  to  relax  completely  before  the  next 
stimulus    reaches    it. 

2.  The  spring  will  now  be  caused  to  oscillate  at  its 
slowest  speed  (10  per  sec),  sliding  clamp  placed  furthest  from 
the    end,    so    as    to    give    a    number    of    stimuli    at    regular 


MUSCLE    FATIGUE.  21  7 

intervals.  As  the  spring  will  give  only  a  limited  number 
of  contacts,  let  your  co-worker  manage  it  whilst  you  attend  to 
the    muscle    record. 

Keep  the  point  off  the  drum  until  ready.  Give  the  sign 
for  the  spring  to  be  set  in  operation,  and  at  once  bring 
the  point  to  bear  upon  the  paper.  Open  S  key  and  close 
it  again  as  soon  as  about  10  contractions  have  occurred. 
When   the    curve    has    fallen,    lift    the    point   off  the    paper. 

The  contractions  will  follow  with  regularity,  and  the  curves 
as  they  succeed  each  other  will  have  the  same  height,  but 
will  be  cut  short  in  their  descent.  The  second  contraction 
of  the  series  may  be  higher  than  the  first,  and  the  remainder 
will    be    of   the   same   height   as   the    second. 

Take  two  more  tracings,  one  with  the  clamp  on  the  spring- 
half  way,  and  the  other  with  the  same  at  the  end  of  its 
slide.  Clamp  firmly  in  position  and  re-adjust  the  needle  each 
time. 

The  contractions  occur  more  frequently  at  each  shortening, 
and  the  third  curve,  and  possibly  the  fourth  may  rise  higher 
than    before,  the   whole   series   being   higher.     Summation. 

3.  Substitute  the  interrupter  for  the  spring,  the  stimuli 
will  be  more  rapid  and  the  curve  will  rise  higher,  be  flat 
topped  and  free  of  signs  of  separate  contractions — complete 
fusion    of    them    having    occurred. 

It  will  not  always  be  necessary  to  use  the  interrupter, 
as  it  depends  upon  the  condition  of  the  frog,  as  to  whether 
so  rapid  a  succession  of  stimuli  will  be  necessary  to  produce 
complete    tetanus. 

Fatigue  of  Muscle.  10 

Required: — Induct orhim,  muscle  chamber  and  stand,  two  Leclanche 
cells,  2  keys  and  5  wires.  Drum  at  a  speed  to  draw  the  muscle 
curve  out  to  2*5 cm,  strong  shocks.     Nerveless  muscle  preparation. 


218  MUSCLE. 

Arrange  the  drum  contact  in  the  P  circuit.  Guide  mark 
on  paper,  S  key  closed  after  ascertaining  that  the  muscle 
responds.     Load   10g. 

As  the  muscle  (gastrocnemius)  is  capable  of  giving  several 
hundred  contractions,  the  gradual  transformation  of  the  muscle 
curve  would  be  obscured  by  the  number  inscribed  on  the 
paper  ;  therefore  only  record    every   25th  or   30th   contraction. 

Adjust  the  recorder  point  to  write  with  an  equable  pressure, 
so  that  there  is  just  enough  play  in  the  point  to  meet  the 
inequalities  of  the  paper  when  the  pillar  handle  is  against 
the    stop.      Start    the    drum. 

After  the  usual  few  preliminary  turns  of  the  drum,  open 
the  S  key  and  apply  the  writing  point  as  the  guide  mark  passes, 
and  lift  it  off  again  just  before  the  end  of  the  revolution. 

Count  from  this  onwards  29  contractions,  and  inscribe  the 
30th,  and   continue  so   doing  until   the  muscle  is  exhausted. 

The  curves  will  gradually  lengthen,  the  chief  change  being 
the  slowing  of  the  relaxation  of  the  muscle.  The  height  will 
fall   until   final   extinction   of    the   contractions   occur. 

To  obtain  a  second   curve  use  the  other  limb   of    the  frog. 

11        Influence   of  the  load  on  the  work  done  by  muscle. 
This  may  be  carried  out  under  the  following  circumstances  : — 

1.   As    a    load    acting    continuously    and    increased 

(a)  By   the    successive   addition    of   separate   weights, 

(b)  By    rapid     increase    in    the    weight,     by    causing    the 

muscle    to    pull    against    a    strong    spring     (isometric 
method). 

Required: — Inductorium,  2  Leclanche  cells,  2  keys,  7  wires,  and 
2  fine,  ten  10«  weights,  muscle  chamber  with  fine  wires  substituted  for 
the  platinum  electrodes.  Nerveless  muscle  preparation.  The  drum  and 
P  key  are  operated  by  hand. 


[NPLUENCB    OF    LOAD.  l' H> 

By   successive   addition   of  separate   weights.       Adjust    the 

muscle    fco    record    a    maximal     contraction.       Take    records    of 

successive    contractions,    moving   the    drum    by    hand    between 

each    for    5mm.      After    the    movement    from    each    contraction 

has    subsided,    turn    the    drum    to    a    fresh    place,    half    a     cm 

from    the    preceding,  add    a    10    gramme   weight,  and    as    soon 

as    the   extension  caused    by  this    is    complete    move   the   drum 

another  half  cm.  stimulate  and  note  that  the  writing  point  on 

extension    of    the    muscle     falls    below    the    point    from    which 

it    started.      Proceed    in    this    manner  until    the    muscle   curves 

are  extinguished.      Compare  the  relative  heights  of  the  various 

contractions,    and     make     an    approximate     calculation    of     the 

work  done  by  the  muscle,  in  gramme  millimetres,  by  measuring 

the  height  of  each  contraction  in  millimetres,  and   multiplying 

in    each    case   by   the    number    of   grammes    lifted.      The  work 

will   rise  with   increase    of    the   weight   up    to    a    certain   point, 

and  will    then    fall. 

Isometric  method.     Contraction  performed   under   a   rapidly 

and    equably    increasing    load. 

Required: — Drum,  muscle  chamber  on  stand,  strong  spring  fixed  to 
a  bar  held  in  a  clamp  upon  the  side  bar  as  in  Fig.  40,  1.  Direct 
stimulation  of  the  muscle  by  drum  contact.  Speed  of  drum  to  draw 
the  muscle  curve  out  to  4:'-3om.  Inductorium,  2  Leclanche  cells,  2  keys, 
7  wires,  and  2  tine. 

In  adjusting  the  muscle  let  it  be  under  slight  tension,  so 
that  there  should  be  no  slack  to  take  up  at  the  first  moment 
of    its    contraction. 

After  the  first  contraction  has  been  recorded,  increase  the 
tension  by  lowering  the  side  bar  2  or  3 mm.  Readjust  the 
writing  point  to  the  original  abscissa.  Repeat  the  process 
after    each    contraction. 

It  will  be  found  that,  within  narrow  limits,  the  contraction 
will    be   increased    by   the   magnification    of    the   load,   and    by 


220 


MUSCLE. 


further     increase    it    is    diminished.       Usually    the    top    of    the 

curve    is    markedly   flattened. 

2.  As  an  afterload.  The  experiment  is  fitted  up  in  the  same 
way  as  the  last,  with  the  exception  that  the  spring  is  detached  from 
the  rod,  and  the  latter  is  employed  as  a  rest  for  the  recorder,  as  in 
Fig.  40,  2.  The  muscle  should  be  extended  without  stretching,  so  that 
the  muscle  may  have  no  slack  to  take  up  at  the  beginning  of  its 
contraction. 


M   Muscle.     B  Side  bar 


Fig.  40. 

1.  Arrangement  for  after-loading.      R  Muscle  recorder. 
C  Clamp  holding  the  supporting  rod. 

2.  For  Isometric  method.     S  Spring  fastened  to  rod  clamped  to  the  side  bar. 


First  load  with  10  grammes,  stimulate,  and  when  the 
relaxation  is  over  after  the  contraction  turn  the  drum  by 
hand    for    half   a    cm. 

Load  with  an  additional  10  grammes  for  each  successive 
contraction   until   all  have  been  added. 

The  curves  as  compared  with  those  obtained  in  the  last 
experiment  are  much  less  in  height  from  the  first,  undergo 
less    increase,   and    subside    sooner. 

The  muscle  is  more  extensible  under  increasing  loads,  and 
consequently  there  is  more  and  more  "  slack  "  to  take  up 
after  each  increment  of  weight  is  added,  hence  the  delay 
in    raising    the    recorder. 


HEATING    AND    COOLING. 


■221 


Effect  of  heating"  and  cooling"  on  the  character  of  the   12 
muscular  contraction. 

Required: — Frog-heart  support  on  the  general  stand,  heating  cylinder 
with  funnel,  and  two  pieces  of  rubber  tubing  25 cm  long,  vulcanite  lid 
for  the  well  of  the  cylinder :  this  has  a  radial  cut  in  it,  so  that 
it  can  be  applied  after  the  muscle  is  fixed  in  position.  Thermometer, 
tin  can  with  spout  for  pouring  water  into  the  funnel,  tin  mug  to  receive 
the  outflow  from  the  cylinder.  Inductorium.  2  keys,  2  Leclanche 
cells,  7  wires,  10 aa  fine  copper  wire  for  attaching  the  femur.  Drum 
contact  in  P  circuit,  and  speed  to  draw  the  curve  out  to  o'm.  S  wires 
attached  as  in  the  figure.  Gastrocnemius  with  a  third  of  the  femur 
attached. 


Fig.  -11.  W  Water  jacket  (cylinder)  upon  G,  the  frog-heart  support.  F  Funnel 
for  inflow  attached  to  the  side  bar  of  the  general  stand.  T  Thermometer.  U  Bent 
bar  for  attachment  of  muscle,  detachable  by  a  side  twist. 

The  femur  is  connected  to  one  side  of  the  inverted  recorder  R  by  a  fine  copper 
wire,  the  writing  point  is  at  the  other  end.  In  the  well  above  G  is  a  glass  cylinder 
for  immersing  the  muscle  in  fluid.     S  Connections  of  S  circuit  for  stimulating. 

Remove  U,  place  the  cylinder  in  position  on  the  frog-heart 
support  (as  shown  in  Fig.  -il),  with  a  little  normal  saline  in 
the   glass,  to   keep    the    muscle   from   drying. 


222  MUSCLE. 

Lay  U  on  a  plate  and  attach  the  muscle  by  its  tendon  to 
the  hook,  fasten  the  fine  copper  wire  to  the  fragment  of  the 
femur  by  wrapping  it  firmly  several  times  round  the  bone 
and  then  twisting  the  wire  upon  itself.  Straighten  the  wire 
in  line  with  the  muscle,  so  that  the  latter  may  pull  directly 
when  attached  to  the  recording  arm  (aluminium). 

Then  holding  U  by  each  end  and  the  copper  wire  so  as  to 
keep  the  muscle  in  position,  replace  and  fix  U  in  the  well  of 
the  cylinder.  Secure  the  free  end  of  the  wire  to  the 
recording  arm,  adjust  the  counterpoise  (10g)  at  R  so  as  to 
take  up  any  slackness  of  the  muscle.  Cover  the  well  with 
the  vulcanite  lid. 

See  that  the  recorder  has  a  fine  point,  and  that  the  latter 
rests  lightly  against  the  drum  surface  when  the  pillar  arm 
touches  the  stop. 

In  carrying  out  the  experiments  attend  to  the  following : — 

(a)  Each  time  a  change  of  temperature  is  established  in  the 
cylinder  ensure  that  the  same  is  effected  in  the  muscle  by 
allowing  one  minute  to  elapse  after  the  mercury  in  the  ther- 
mometer comes  to  rest. 

(b)  Mark  the  temperature  against  each  curve  as  soon  as 
it    has    been    drawn. 

(c)  Observe  in  each  case  changes  in  the  height  and  in  the 
duration  of  the  muscle  curve,  and  note  in  the  latter  case  if 
the   contraction   or  the  relaxation  is  most  affected. 

(d)  If  the  temperature  of  the  muscle  be  too  suddenly  and 
largely  changed,  the  muscle  may  pass  into  continued  twitch- 
ings  ;  these  will  pass  off.  The  suddenness  of  the  change  acts 
as   a   stimulus. 

1.  Take  a  tracing  at  the  temperature  of  the  room  as 
a   control   experiment. 


CURARA    EXPERIMENT. 


223 


2.  Cool  the  muscle  by  filling  the  cylinder  with  water  at 
the  required  temperature.  Take  successive  tracings  at  8°,  6% 
4°,  and   2°   C. 

3.  Warm  the  muscle,  taking  tracings  at  every  3°  C  rise, 
above  10"  C.  Inscribe  these  curves  upon  a  portion  of  the 
recording  surface  immediately  below   the  record   of    No.   2. 

Curara  experiment.     Direct  excitability  of  muscle. 

Required: — No  recording.  Inductorium,  2  Leclanche  cells,  2  keys, 
3  ordinary  and  2  thin  wires,  hand  electrodes,  waxed  paper,  narrow  tape 

and  a  1  •"'    solution  of  curara  in  water  (filtered). 


Fig.  42.  P  Poisoned  limb.  L  .Seat  of  ligature.  W  Waxed  paper  under  the 
nerves.  C  Commutator,  less  cross  wires ;  wired  to  S  circuit,  outleads  M  to  muscle, 
H  to  hand  electrodes. 

Decerebrate  the  frog  and  plug  the  opening  into  the  skull 
firmly  to  prevent  bleeding.  Having  ligatured  the  middle  of 
one  thigh  firmly  with  tape  (unpoisoned  limb),  so  as  to  stop 
the  circulation,  but  not  to  crush  the  nerve,  inject  5  drops 
of  curara  solution  into  the  dorsal  lymph  sac,  half  or  three- 
quarters  of  an  hour  before  required.  Wait  until  the  poison 
has  produced  its  effect,  i.e.,  until  reflexes  excited  in  the 
poisoned    limb    are    absent. 

Expose  both  sciatic  nerves  throughout  their  whole  lengths, 
and  remove  the  urostyle ;  carefully  introduce  beneath  both  of 
them   as   high    up    near   the   spinal  column   as   possible    a   piece 


13 


224  MUSCLE. 

of  waxed  paper,  upon  this  place  the  nerves  on  the  hand 
electrodes. 

Fix  the  knees  with  pins  passed  through  the  quadriceps 
tendon,  and  turn  the  feet  out  so  that  their  movements  may  be 
well    seen   when    the    calf   muscles    contract. 

Expose  the  calf  muscles  on  both  sides  and  attach  the 
thin   wires,  one   to    each    tendon. 

Carry  the  connection  of  the  hand  electrodes  and  the  thin 
wires  to  opposite  sides  of  the  commutator  (less  cross  wires), 
wire    the    inleading    terminals    to    the   S    circuit. 

Induction  shocks  can  now  be  thrown  into  either  the  muscles 
or  the  nerves. 

First  stimulate  the  nerves  with  minimal  interrupter  shocks, 
and  note  that  the  muscle  of  the  poisoned  side  does  not 
contract,    whilst    the    other    does. 

The  nerve  has  not  been  paralysed,  because  you  are  stimulating 
a  portion  of  nerve  which  has  been  under  the  influence  of  the 
poison. 

Next  stimulate  the  muscles,  commencing  with  minimal 
shocks  and  note  which  muscle  contracts  first  ;  usually  the 
unpoisoned  one  does  (Rosenthal  effect).  This  is  due  to  the 
excitability  of  the  nerve  in  the  muscle. 

Both  muscles  respond,    hence  they  are  not  paralysed. 

There  remains  the  conclusion  that  the  end  plates  of  the 
nerves  are  the   seat  of  the   change. 

14   Effect   of  Veratria  upon   muscle. 

Required: — Recording  and  stimulating  arrangements  as  for  single 
contraction    with    direct    stimulation   of    muscle. 

Apply  a  ligature  to  one  leg  of  the  frog  in  the  same  manner  as  for 
the  curara  experiment,  and  inject  10  drops  of  1/1000  solution  of 
sulphate  of  veratria  into  the  dorsal  lymph  sac.  The  full  effect  of  the 
drug  will  take  three-quarters  of  an  hour  to  develop.  When  stimulated 
the  poisoned  limb  will  exhibit  prolonged  contractions. 


EFFECT    OF    VERATRIA    UPON    MUSCLE.  225 

Prepare  both  nerve  muscle  preparations  and  take  successively 
records  of  the  unpoisoned  and  poisoned  muscles. 

If  the  action  of  the  drug  is  well  developed,  the  muscular 
contraction  will  extend  several  times  round  the  drum  when 
the  latter  is  at  a  speed  which  draws  the  normal  contraction 
out   to   5cm- 


15 


CHAPTER    XXXII. 


EXPERIMENTS    ON    NERVES. 


Rate  of  nerve  conduction,  e.g.,  the  velocity  with  which 
a    nervous    impulse    travels    along    a    nerve. 

Required : — Inductormm,  2  keys,  2  Leclanche  cells,  commutator  less 
•cross  wires,  double  electrodes,  electric  style,  and  12  wires.  Muscle 
chamber,  with  fine  writing  point.  Nerve  muscle  preparation,  with 
nerve  most  carefully  dissected  and  with  piece  of  spinal  column  attached. 
Drum   at   very   fast   speed. 

Set    up    the    P    circuit    with    2    cells    and    in    drum    contact. 
Wire    the    S    circuit    from    S    key    to    the    inleading    binding 

screws    of   a    Pohl's   commutator,    less    cross    wires. 

The  two  sets  of  outleading  terminals  of  the  Pohl  are  wired 

to   the   two  sets  of    external   terminals  of    the  muscle-chamber. 

From  the  latter  remove 
the  platinum  and  non- 
polarisable  electrodes  and 
in  their  stead  attach  the 
double  electrodes  Fig.  43, 
so  that  the  two  sets  of 
wires  shall  be  connected 
respectively  with  opposite 
sides  of  the  commutator. 
When  the  rocker  lies  one 
way  it  will  lead  the  stimu- 
lating current  into  wires 
at   a    near  the  muscle,  and 

when  turned  over  it  will   establish    connection  with   b  farthest 

from    the    muscle. 

Set  up  the  style  to  write  9Gth  seconds  under  the  muscle  curve. 


FIG.  43.  M  Muscle.  N  Nerve  on  G,  glass- 
plate  across  this,  a  and  b,  pairs  of  wires  at 
2-5  cm  interval  ending  in  A,  B,  for  attachment 
to  binding  screws  of  muscle  chamber.  L  Lead 
plate. 


RATE  OF  NERVE  CONDUCTION.  227 

Clamp  the  nerve  muscle  preparation  into  position  and  lay 
the  nerve  across  the  electrodes  (2*5 cnL)  as  in  the  Figure.  If 
very  long  dispose  it  in  a  curve,  and  its  length  can  be  sub- 
sequently   measured. 

All  being  ready  and  the  stimulus  to  the  nerve  having  been 
tested   in  both  positions  of    the  commutator, 

Perform    the    experiment    as    follows  : — 

1.  Keeping  the  S  key  closed — start  the  drum — apply  the 
style  to  the  paper,  the  rate  should  be  such  as  to  draw 
the  96th  second  trace  interval  to  not  less  than  1  cm\  Adjust 
the  speed  accordingly.  Make  this  trial  at  the  bottom  of  the 
paper. 

2.  Apply  the  muscle  recorder  and  style  to  the  paper  over- 
lap and  adjust  them  to  write  above  each  other,  lift  them 
off.     Mark   the   paper  over-lap. 

Start  the  drum  and  inscribe  an  abscissa  and  time  record 
during  one  revolution  of  the  drum,  being  careful  to  remove 
the  points  before  the  next  revolution  begins. 

Let  the  drum  continue  to  revolve  from  this  onwards  until 
the  experiment  is   complete. 

3.  Two  successive  muscle  tracings  will  now  be  taken  on 
immediately  succeeding  revolutions  of  the  drum,  the  one  as 
the  result  of  stimulating  at  a,  and  the  other  at  b.  This  is 
materially  facilitated  if  your  co-worker  takes  charge  of  the 
commutator,  and  turns  it  over  the  moment  you  give  the 
signal    that    the    first    record    is   complete. 

Lift  off  the  points  the  moment  the  second  record  has  been 
taken,  and   stop   the   drum. 

Next  measure  the  interval  between  the  two  muscular  con- 
tractions. This  interval  is  best  measured  about  midway  in 
the  ascent  of   the  curves  where  thev  are  clear  of   each  other. 


228  NERVE. 

Write  a  second  abscissa  through  the  position  chosen,  and 
measure  the  distance  between  them  against  the  time  tracing 
immediately  below. 

From  this  calculate  the  rate  in  metres  per  second  as 
follows : — 

The  interval  is  estimated  as  lying  between  1/8  to  a  1/10 
of  a   1/96   of   a   second   time  interval.      Assume  it  to  be   1/9. 

The  nervous  impulse  has  therefore  taken  1/864  of  a  second 
to   travel    2-5 cm. 

In  1  second  the  impulse  would  travel  864  x  2*5  cm  =  2160  cm 
=   21*6    metres.     A  somewhat  low  estimate. 

Rate  of  transmission  of  a  nervous  impulse,  measured 
by  means  of  the  pendulum  myograph  and  the  electrical  method. 

Required: — Electric  style  fastened  to  the  side  bar  of  th^  general 
stand,  muscle  break,  2  commutators,  inductorium,  2  keys,  4  Daniell 
cells,  and    16   wires. 

All  the  records  are  made  in  this  experiment  with  one  time 
marker,  which  is  so  connected  to  the  time  distribution  board 
and  the  muscle  break  by  means  of  a  commutator  that  it 
can  be  thrown  into  line  with  either  of  these  as  required. 
The  movement  of  the  muscle  consequent  upon  the  stimulation 
of  the  nerve  is  recorded  by  the  break  of  the  circuit.  This 
circuit  whilst  closed  keeps  the  point  of  the  style  depressed, 
on  breaking  the  point  flies  up,  making  a  sharply  defined 
upward  stroke,  which  is  more  easily  read  than  the  muscle  curve 
in  the  previous  experiment. 
Set  up  as  follows  (Fig.  44)  : — 

1.  Stimulating  circuit.  The  board  of  the  pendulum  carries 
a  projecting  arm  which,  in  swinging  past,  knocks  open  a  closed 
key.  This  key  is  fixed  to  the  floor  of  the  instrument  in 
such  a  position  that  the  pendulum  is  travelling  nearly  at  its 
maximum  speed   when  it  opens  the  key. 


RATE    OF    NERVE    CONDUCTION. 


229 


This  key  is  included  in  the  P  circuit  of  the  inductorium 
(2  cells).  The  S  circuit  is  wired  to  the  inleading  terminals 
of  the  commutator  (less  cross  wires).  The  opposite  leading 
out  pairs  of  terminals  are  wired  respectively  to  the  upper 
and  lower  terminals  of  the  double  electrodes  of  the  muscle 
break.     These  electrodes  are  2'5cm   apart. 


Fig.  44.  1  Pendulum.  2,  2,  Catches.  X  Smoked  paper  and  points  to  style. 
3  Plummet  for  comparing  inscriptions,  hooked  aside  when  not  in  use,  to  the  right 
knock-open  key  in  P  circuit  4.  S  Circuit  by  5  to  Pohl  6,  wired  to  double  electrodes 
in  muscle  break  7.  In  latter,  muscle  is  attached  to  key  in  circuit  8  to  Pohl  9,  which 
latter  is  wired  to  style  X,  and  time  board  10. 


2.  Recording  circuit.  Wire  the  electric  style  to  the  inlead- 
ing terminals  of  a  second  commutator  (less  cross  wires)  and 
one    pair    of    the    outleading   ones    to    the   aluminium    key   of 


2-30  NERVE. 

he  muscle  break,  including  in  this  circuit  2  cells;  the  other 
outleading  pair  wire  to  the  time  board  terminals  on  the 
right    of    the    pendulum    case. 

See  that  all  connections  are  perfect. 

To  the  pendulum  board,  which  is  of  wood,  paper  can  be 
pinned.  The  latter  is  smoked  on  a  drum  and  when  cool  is 
cut  off  and  will  then  remain  flat.  Trim  to  the  required  size 
and  fasten  in  position  with  drawing  pins. 

It  next  receives  the  time  mark.  The  pendulum  being  held 
to  the  right  by  the  catch,  a  vertical  line  is  marked  upon  it 
at  its  left  extremity  by  flicking  the  plum  line  (which  hangs 
from  the  centre  of  oscillation)  against  it  as  a  guide  for  the 
application    of    the    writing    points. 

The  electric  style  is  set  to  this  line,  and  whilst  it  is 
repeating  its  96  vibrations  a  second  the  pendulum  is  released, 
swings  past,  and  the  time  subdivision  is  accomplished.  Raise 
the  writing  point  off  the  surface. 

As  the  pendulum  always  starts  from  the  same  point  it 
practically  always  swings  at  the  same  rate,  and  the  one  time 
mark  is  sufficient  for  all  the  records  which  may  be  inscribed 
above  each  other  upon  the  same  paper  as  long  as  they  all  start 
from  the  plumb  line.  They  will  also  have  the  same  relation- 
ship to  the  time  at  which  the  knock-open  key  is  actuated. 

The  style  is  now  connected  to  the  muscle  break  key  by 
turning  the   rocker  of   commutator  9   (Fig.   44). 

Close  the  aluminium  key  by  adjusting  the  screw  counter- 
poise ;   and  see  that  the  muscle  is   slightly  stretched. 

Set  the  style  close  above  the  time  record,  close  the  knock- 
open  key,  turn  commutator  6  to  the  electrodes  nearest  the 
muscle,  open  the  S  key  and   release   the  pendulum. 

Raise  the  point  off  the  writiny  surface,  return  the  pendulum 
to    the   right-hand   catch,   close   the    knock-open    key   and    take 


KLKCTROTONUS. 


231 


a  second   record,   this   time   with  commutator    6   turned    to   the 
more  distant  electrode. 

Read  off  the  difference  between  the  two — this  will  give  the 
time  consumed  by  the  nervous  impulse  in  travelling  from  the 
one  point  of  stimulation  to  the  other.  Calculate  the  rate  in 
metres  per  second  as  before. 

Electrotonus.     Electrotonic  variation  of  nervous  excitability,    yj 

Required: — Inductorium  for  single  induction  shocks,  two  Daniell 
cells,  Pohl's  commutator,  muscle  chamber  with  non  -  polarisable  and 
platinum  electrodes,   11  wires,  3  keys.     Nerve  muscle  preparation. 


E     N 


Fig.  45.  Electrotonus.  Position  of  keys,  &c,  on  the  table.  Stimulating  circuit 
P,  S  to  E  the  platinum  electrodes  near  the  muscle  M.  Polarising  key  K.  C  com- 
mutator.    N  non-polarisable  electrodes  on  nerve  above  E.     L  Leclanch£  cells. 


Set  up  as  follows   (Fig.   45)  : — 

(a)  The  stimulating  circuit.  Connect  the  S  circuit  to  the 
binding  screws  of  the  platinum  electrodes  on  the  outside  of 
the  muscle  chamber. 

(b)  Polarising  circuit.  Arrange  as  in  the  diagram.  Connect 
the  positive  pole  of  the  battery  to  the  key  (break)  and  wire 
the  poles  to  the  commutator  C,  and  the  pair  of  terminals 
nearest  the  muscle  chamber  to  the  terminals  of  the  non- 
polarisable  electrodes   of   the  latter. 


232  NERVE. 

Examine  PohPs  commutator,  Fig.  23.  Take  care  in  attach- 
ing the  wires  to  make  good  contacts,  also  that  there  is 
sufficient  mercury  in  the  cups  of  the  commutator  to  cover  the 
ends  of  the  rocker  arms  and  those  of  the  binding  screws  that 
lead  into  the  former. 

Prepare  non-polarisable  electrodes: — Take  a  large  teaspoonful 
of  China  clay  (Kaolin)  and  work  it  into  a  stiff  paste  with  a 
little  normal  saline.  Perform  the  operation  upon  a  clean 
plate,  and  with  a  clean  porcelain  or  horn  spatula,  avoiding  all 
contact  with  the  fingers.  Remove  the  glass  tubes  from  their 
holders  in  the  muscle  chamber,  wash  them  clean  with  warm 
water  and  dry  them,  then  stop  the  bevelled  end  of  each  with 
the  clay  for  1  cm,  pressing  it  in  compactly  with  the  spatula. 
Adapt  the  end  to  receive  the  nerve  by  moulding  the  clay  into 
a  central  ridge  lengthways  over  the  bevelled  end. 

Replace  in  the  holders  and  adjust  them  close  to  each  other 
and  as  near  to  the  platinum  electrodes  as  possible. 

Proceed  as  follows  : — 

Influence  of  the  positive  pole  (anode).  Turn  the  commutator 
rocker  so  that  the  positive  pole  leads  to  the  non-polarising 
electrode  nearest  the  muscle.     Keep  key  K  open. 

2. — Find  the  minimal  opening  induction  shock  which  will 
suffice  to  cause  a  muscular  contraction. 

3. — Close  the  polarising  circuit  and  stimulate  the  nerve  with 
the  opening  S  shock — there  should  be  no  contraction.  The 
production  of  the  anelectrotonic  condition  (plus  pole)  has 
lowered  the  excitability  of  the  nerve,  and  the  stimulus  is  now 
insufficiently  strong  to  excite  it. 

Influence  of  the  negative  pole  (anode).      Turn  the  commutator 
so  as  to  substitute  the  minus  for  the  plus  pole. 
Keep  key  K   open. 


pfluger's  law.  233 

Find  the  induction  shock  which  is  just  too  weak  (subminimal) 

to  stimulate  the  nerve. 

Close  the  polarising  circuit  and  stimulate —the  muscle  will 
contract.  The  influence  of  the  kathode  has  raised  the  excita- 
bility of  the  nerve,  so  that  the  previously  inefficient  stimulus 
is  now  sufficient  to  excite  it. 

Instead  of  using  induction  shocks,  the  nerve  may  be  continu- 
ously stimulated  by  applying  a  drop  of  strong  salt  solution 
to  the  nerve  in  the  same  position,  producing  "salt  tetanus." 
A  record  can  be  taken,  and  the  influence  of  the  plus  and 
minus  poles  is  indicated  by  changes  in  the  height  of  the 
contraction. 

Pfliigers   Law.     Polar  excitation   of  nerve.  18 

Required: — Rheocord,  commutator,  1  key,  7  wires,  and  4  or  5  cells 
(Leclanche  or  dry  cells).     Muscle  chamber  on  stand  and  drum. 

The  Rheocord  (Burdon  Sanderson's  pattern)  consists  of  a  platinum 
iridium  wire  of  about  20  ohms  resistance,  arranged  for  campactness  in 
zig-zag  upon  a  board,  and  ending  in  screw-down  terminals  at  each  end. 
A  movable  block  B,  with  terminal,  is  for  the  purpose  of  establishing 
contact  at  any  point  with  the  wire.  The  appliance  is  used  in  short 
circuit  in  this  experiment,  and  in  such  a  manner,  see  Fig.  46,  as  to 
vary  the  potential  at  the  non-polarisable  electrodes. 

Owing  to  the  large  resistance  of  the  nerve,  lcm  of  which  is  ap- 
proximately equal  to  80,000  ohms  and  non-polarisable  electrodes  700 
ohms  each,  very  little  actual  current  passes,  and  it  becomes  a  question 
of  polarity. 

The  copper  leads  are  of  insignificant  resistance.  When  the  rheocord 
is  out  of  circuit  (with  block  off),  the  potential  at  the  clay  electrodes 
is  little  less  than  that  at  the  poles  of   the  battery. 

As  soon  as  the  rheocord  is  introduced,  a  great  drop  of  potential 
occurs  at  the  electrodes. 

Set  up  the  connections  as  shown  in  Fig.  46.  Mark  the 
position  of  the  rocker  of  the  commutator  when  the  plus  pole 
is  at  the  non-polarisable  electrode  nearest  the  spine  (descending- 
current).  On  reversal  the  plus  pole  will  be  nearest  the  muscle 
(ascending   current). 


234 


NERVE. 


The  potential  at  the  electrodes  on  the  nerve  is  varied  by 
short-circuiting  through  the  derivation  circuit  formed  by  the 
rheocord. 

Three  strengths  of  potential  will  be  sought  for  in  order  to 
obtain    the    following    result. 

The    following    is    given    as    an    approximate    guide  : — 

1.  "Weak  : — One  cell  with  the  block  of  the  rheocord  close 
to    the   return    terminal. 


Fig.  46.  Pfliiger's  Law.  L  Cells.  K  Key.  B  Movable  block  off  R,  short  circuits 
when  placed  on  rheocord  wire  R.  C  Commutator.  N  Non-polarisable  electrodes  on 
the  nerve.     M  Muscle. 


2.  Medium  : — One    or    two    cells    with    the    block    on    the 
second  or  third   stretch  of   the  wire  from  the  return  terminal. 

3.  Strong  : — Three    or    four    cells    with    the    block    off    the 
rheocord. 

Modify    the   current    so   as    to   obtain   the    following   results  : — 


Weak 

Medium 

Strong 

Explanation  : 


Ascending  current. 
.Make.  Break. 

C       ...      — 


Descending  current. 
Make.  Break. 

C       ...      — 


c 


c 
c 


c 
c 


c 


Excitation    is    either    due    to    production    of 
katelectrotonus    or    to    disappearance   of   anelectrotonus. 
The    former    is    the    more    efficient    stimulus. 


pfluger's  law.  235 

With  a  weak  current  the  kathode  is  alone  operative.  With 
a  medium  one  both  are  operative  as  stimuli,  whilst  at  the 
same  time  the  anode  is  not  powerful  enough  to  block  the  ex- 
citation due  to  the  katelectrotonic  stimulus.  With  the  strong 
ascending  C,  the  anode  blocks  at  make,  but  stimulates  by  its 
fall  at  break. 

In  the  case  of  the  strong  descending,  the  kathode  has 
nothing  to  block  its  action  at  make,  but  at  break  the  fall 
of  excitability  on  the  disappearance  of  katelectrotonus  is 
held   to  block   the  stimulus  due    to  the  fall   of    anelectrotonus. 


CHAPTER    XXXIII. 

ELECTRICAL   MANIFESTATIONS    IN   MUSCLE 
AND   NERVE. 

Demarcation  and  action  currents. 

Required: — Astatic  galvanometer  (permanently  set  up  and  adjusted 
on  a  stone  shelf  on  the  wall  of  the  dark  room).  Non-polarisable  electrodes 
on  movable  supports.  Shunt  or  resistance.  Block  of  paraffin.  Inductorium 
and  platinum  electrodes  on  stand.     Nerve  muscle  preparation. 

Set  up  as  follows  : — Wire  the  non-polarisable  electrodes  to 
the  shunt  or  resistance  in  short  circuit  and  thence  to  the 
galvanometer.  Observe  the  notice  that  if  the  north  binding- 
screw  of  the  galvanometer  be  positive,  the  spot  of  light  will 
be  deflected  to  the  left,  and  vice-versa  (Fig.  47). 

Support  the  nerve  muscle  on  the  block  of  paraffin  and  adjust 
the  nerve  across  the  platinum  electrodes  and  connect  the  latter 
with  the  circuit  of  the  S  inductorium.  The  last  should  be 
placed  not  less  than  three  feet  from  the  galvanometer. 

Light  the  lamp  and  place  it  so  that  the  reflected  spot  of  light 
.shall  be  as  brilliant  as  possible  with  the  vertical  wire  in  focus. 

Test  the  electrodes  to  see  if  they  yield  a  current.  Place 
their  points  in  contact,  and  close  the  circuit  through  the 
galvanometer.  If  they  do,  the  spot  of  light  will  travel  away 
from  the  middle  of  the  scale  to  one  side  or  the  other.  Note 
the  amount  of  the  deflection,  and  if  it  be  more  than  a  couple 
of  cm,  fresh  electrodes  must  be  prepared.  If  slight,  note  its 
amount  and  direction. 

Place  one  electrode  in  contact  with  the  centre  of  the  surface 
of    the    muscle,    and    the    other    near    the    tendon.      Close    the 


CURRENTS  IN  MUSCLE  AND  NERVE,  237 

galvanometer  circuit.  Note,  from  the  direction  in  which  the 
spot  moves,  which  of  the  two  points  of  the  muscle  touched 
by    the    electrodes    is    positive    to    the    other. 

Now  stimulate  the  nerve  with  interrupter  shocks  of  just 
sufficient  strength  to  tetanise  the  muscle,  and  observe  that 
the  previous  deflection  is  diminished.  The  action  current  is 
opposed    in    direction    to    the    demarcation    current. 

Next  cut  off  the  end  of  the  muscle  near  the  tendon,  and 
apply  the  electrode  to  the  artificial  cross  section  ;  the  deflection 
may  be  so  great  as  to  throw  the  spot  off  the  scale.  Bring 
it  back  by  putting  a  plug  into  the  shunt  so  that  only 
a  fraction,  i.e.,  1  10,  1/100,  or  1  1,000,  passes  to  the  galvano- 
meter.     The    shunt    makes    a    proportionate    short    circuit. 

Again  stimulate  the  nerve,  the  movement  of  the  spot  in 
the    contrary   direction   will    be    more    marked. 

Currents  in  the  frog's  heart.  Excise  the  entire  heart,  in- 
cluding the  sinus,  dispose  the  electrodes  close  to  each  other 
so  that  the  heart  may  rest  with  its  base  upon  one  of  them, 
and    the    apex    on    the    other. 

Close  the  galvanometer  circuit  between  the  beats  and  observe 
that  there  is  a  deflection  (demarcation  current),  which  under- 
goes a  sudden  diminution  at  each  contraction  (action  current). 

Current  from  the  nerve.  Remove  the  two  sciatic  nerves, 
cut  them  as  long  as  possible,  and  arrange  them  across  one 
electrode  with  both  ends  of  each  hanging  down  and  touching 
the    second   electrode. 

Close  the  galvanometer  circuit  and  note  the  deflection. 
The  longitudinal  surfaces  of  the  nerves  are  positive  to 
their    ends. 

The  cut  end  of  a  nerve  is  in  a  state  of  katelectrotonus, 
due  to  changes    accompanying    the    death    of    the    nerve. 

Capillary  electrometer  (Fig.  47,  2).  Repeat  the  previous  experi- 
ment  with  this  instrument  instead  of  the  galvanometer. 


238 


TISSUE    CURRENTS. 


Requirements     the     same    as     before,    and    couple    the    wires 
from    the   electrodes    to    the    terminals    of   the   electrometer. 


The    latter   is    set   ujd    on    a 
dark    room,    and    consists    of    a 


bracket   on    the    wall    of    the 
glass     tube    drawn    to    a    fine 


Fig.  47.  G  Galvanometer.  L  Lamp  and  scale.  H  Shunt.  C  Commutator  with 
leading-in  terminals  in  circuit,  with  E  Non-polarisable  electrodes.  P  Paraffin  block 
supporting  a  muscle,  the  nerve  of  which  rests  on  platinum  electrodes  S  connected 
to  S  circuit  of  inductorium. 

1.  Non-polarisable  electrode.  C  China  clay  point  on  glass  tube  S  containing 
saturate  zinc  sulphate  solution.     Z  Zinc  rod  (amalgamated)  corked  into  place. 

2.  Diagram  of  capillary  electrometer.  C  Glass  tube  with  capillary  end,  con- 
taining mercury  (black),  dipping  into  1  in  10  sulphuric  acid  A.  E  Wires  of  platinum 
in  the  electrometer,  attached  to  "leading- off"  electrodes.  There  is  an  arrangement 
for  exerting  pneumatic  pressure  on  the  mercury  in  C  to  adjust  its  position  in  the 
capillary.  The  mercury  moves  in  the  direction  of  the  negative  pole.  The  electro- 
meter is  substituted  for  the  galvanometer  and  shunt  in  the  first  arrangement  by 
attaching  its  wires  to  the  commutator  C. 

capillary  end,  in  which  there  is  mercury,  and  which  dips 
into  a  small  trough  containing  10  p-c-  H2S04.  There  is  a 
small  pool  of  mercury  in  the  latter  also.  The  terminal  wires 
dip  into  the  mercury  in  each  case. 


CAPILLARY    ELECTROMETER.  239 

The  position  of  the  thread  of  mercury  in  the  capillary 
is  controlled  pneumatically  by  means  of  a  small  mercury 
pressure  apparatus.  The  meniscus  of  the  mercury  is  observed 
through    a    microscope    with    a    power    of    100    diameters. 

The  terminals  of  the  electrometer  are  provided  with  a 
short-circuiting  key,  which  is  to  be  kept  closed  when  the 
instrument    is    not    in    actual    use. 

The  less  the  diameter  and  length  of  the  capillary,  so  much 
the  greater  will  be  the  sensitiveness  of    the  instrument. 

Only  small  electrical  pressures  may  be  exerted  upon  the 
mercury  in  the  capillary,  and  an  ordinary  cell  must  on  no 
account  be  placed  in  its  circuit  unless  high  resistances  are 
included  at  the  same  time,  as  bubbles  of  H  gas  and  crystals 
of  mercury  sulphate  will  separate  with  the  current  from  a 
single  Leclanche,  thus  rendering  the  capillary  useless. 

The  movements  of  the  meniscus  are  sudden  and  dead  beat, 
and  the  mercury  always  moves  away  from  the  plus  pole  in 
proportion  to  the  electrical  pressure  exerted. 

The  instrument  is  used  as  a  pressure  detector  and  not  for 
measuring  current,  and  the  quickness  of  its  response  makes  it 
the  only  means  for  detecting  small  and  rapid  variations  in  the 
electrical  condition  of  animal  tissues. 

The  condition  and  changes  of  potential  already  referred  to  in 
the  previous    exercise  are  readily  confirmed  by  its  aid. 


CHAPTER    XXXIV. 
ON    THE    CIRCULATORY    SYSTEM. 

Beat  and  sounds  of  the  human  heart. 

Feel  the  apex  beat.  Place  the  points  of  the  fingers  of  your 
hand  upon  the  fifth  intercostal  space  on  the  left  of  the  sternum 
and  feel  the  movements  of  the  heart. 

Count  the  rate  of  pulsation  in  the  minute  by  your 
watch. 

Note  the  character  of  the  impulse  and  its  variations  with 
the  respiration  and  the  posture  of  the  body — sitting,  standing, 
and  in  the  reclining  position.  Count  the  number  to  each 
respiration. 

2.  Listen  to  the  sounds  of  the  heart  with  a  stethoscope  (binoral). 
The  phonendoscope  is  the  best  form  of  this  instrument.  It 
consists  of  a  heavy  metal  disc  excavated  upon  one  side,  over 
this  a  vulcanite  diaphragm  is  fixed.  Two  rubber  tubes  are 
attached  to  the  other  side  by  short  metal  tubes,  and  their 
free  ends  by  ear-pieces  to  each  ear.  One  hand  holds  the 
instrument. 

Seat  yourself  opposite  the  subject  and  hold  the  diaphragm 
against  the  surface  of  the  chest,  a  little  to  the  sternal  side  of 
the  apex  beat  and  clear  of  clothing,  which  must  not  be  allowed 
to  rub  against  the  instrument. 

Distinguish  between  the  two  sounds.  The  longer  one  corre- 
sponds to  the  ventricular  systole,  the  sharp  second  sound  to  the 
closure  of  the  aortic  and  pulmonary  valves. 


CARDIOGRAM.  241 

3.  Trace  a  cardiogram.  The  cardiograph  consists  of  two 
pneumatic  tambours  connected  by  an  india-rubber  tube. 

The  tambour  for  the  heart  is  provided  on  its  rubber  mem- 
brane with  an  ebonite  button,  the  capsule  rests  on  the  chest 
wall  and   is   secured  in  position   by  an  elastic  girth. 

The  recording  tambour  carries  a  recorder  for  writing  on  a 
smoked  surface. 

The  tube  which  connects  the  two  is  provided  with  a  brass 
valve  to  regulate  the  air  tension  in  the  system. 

Place  the  button  of  the  heart  tambour  over  the  apex  beat 
on  the  bared  chest  of  the  subject,  who  is  seated  on  a  chair. 

Let  the  drum  revolve  at  a  medium  speed,  and  apply  the 
recorder  to  its  surface  and  take  half-a-dozen  curves.  Adjust 
to  get  a  maximum  tracing. 

The  curve  will  present  four  well-marked  features  : — 

(a)  A  slow  rise — the  heart  is  distending — immediately  followed 
by 

(b)  A  rapid  rise — -the  ventricular  systole — which    passes  into* 

(c)  An  irregular  plateau,  at  the  end  of  which  occurs  the 
closure  of  the  semi-lunar  valves. 

(d)  A  descent — the  heart  loses  redundancy.     Diastole. 

Next  repeat  the  cardiogram,  together  with  a  pulse  tracing- 
written  below  it.  Use  the  sphygmographic  tambour  belonging 
to  a  Brondgest  pantagraph,  and  adjust  it  to  the  wrist  over 
the  radial  pulse.  Fix  into  place  with  tapes.  The  wrist  must 
be  supported  on  a  properly  shaped  pad  in  a  slightly  over- 
extended position.  The  arm  must  be  unconstrained  and  the 
muscles  relaxed.  Adjust  the  pressure  of  the  tambour  button 
by  means  of  the  screw  which  regulates  the  tension  of  the 
spring  until  the  maximal  excursion  of  the  recorder  is  produced. 

Let  both  points  write  vertically  beneath  each  other, 


242  CIRCULATION. 

Inscribe  a  1/10  second  trace  below  them. 

Take  a  record  for  one  revolution  of  the  drum  only  at  a 
time. 

Note  carefully  the  relative  position  of  the  dicrotic  wave  in 
the  pulse  curve  to  the  end  of  the  systolic  plateau  of  the 
cardiogram,  and  estimate  the  time  interval  by  which  they  are 
separated.  This  interval  corresponds  to  the  time  which  the 
pulse  wave  has  taken  to  travel  from  the  aortic  valves  to  the 
wrist.  Measure  approximately  this  distance  in  centimetres  by 
means  of  a  tape  measure,  and  from  these  data  calculate  the 
rate  of  propagation  of  the   pulse  wave  per  second. 

The  length  of  the  pulse  wave  can  be  determined  by  multi- 
plying its  rate  of  propagation  per  second  by  the  time  which 
it  takes  to  pass  a  given  point,  e.g.,  the  time  which  it  takes  to 
pass  the  point  at  the  wrist  to  which  the  sphygmograph  has 
been  applied. 

21        The  pulse  tracing",  with  Dudgeon's  sphygmograph. 

Place  the   wrist  of  the  subject  in  position   upon  the  pad. 

Mark  the  point  over  the  radial  where  the  pulse  is  most 
distinctly  felt,  and  rest  the  button  of  the  spring  of  the 
sphygmograph  upon  the  chosen  point,  and  secure  in  position 
with  the  tape. 

Adjust  the  pressure  of  the  button  upon  the  artery  by 
means  of  the  eccentric  until  a  maximal  excursion  of  the 
recorder  is  obtained. 

Wind  up  the  clockwork,  insert  a  strip  of  smoked  paper 
between  the  guide  wheels,  and  let  the  paper  travel  past  the 
recording  point  as  soon  as  the  latter  moves  regularly. 

Examine  the  tracing  : — The  first  part  of  the  curve  is  a 
main  upstroke,  devoid  of  secondary  oscillations,  upon  the 
descent  is  the  dicrotic  wave,  preceded  by  the  corresponding 
notch. 


HLOOD    PRESSURE. 


243 


The  other  features  consist  of  secondary  oscillations,  which 
vary  according  to  the  tension  of  the  arterial  wall  and  the 
inertia  of  the  instrument. 

A  hard  pidse  shows  many  of  these,  owing  to  the  quick 
elastic  reactions  which  accompany  the  greater  state  of  tension. 

A  soft  pulse,  on  the  other  hand,  shows  less  height  of  curves, 
less   acuteness   in   the  angles,  and  a   less   number  of   secondary 

oscillations. 

Blood  Pressure.  Estimate  this  by  means  of  the  sphyg- 
mometer (Hill  &   Barnard). 

See    that    the    column    of    fluid    stands    at    zero.      To    adjust 
this,   hold   the  instrument  vertically,   open  the  tap  at   the  top, 
and    press   the    rubber   ampulla    gently    upon    the   surface   of   a 
table,  supporting  it  with 
both     hands     to     control 
your    movement,    and 
closing     the     tap     the 
moment  the  fluid  stands 
at  zero. 

(a)  Artery.  Press  the 
ampulla  upon  the  radial 
until  the  maximum  pul- 
sation of  the  column  of 
fluid  is  obtained.  The 
pressure  will  then  be  the 
same  inside  and  outside 
the    vessel,    e.g.,    in    the 

sphygmometer,     and     its 

Fig.  48.     Bill  A:  Barnard's  Sphygmometer, 
magnitude     is     read     off 

on    the    stem,  which    is    graduated    in    terms   of    millimetres   of 
mercury. 


244 


CIRCULATION. 


(b)  Vein  ;  choose  one  of  the  large  veins  on  the  back  of  the 
hand,  and  find  the  positions  of  two  valves  in  its  course. 
Place  the  hand  flat  on  a  chair  close  to  that  upon  which  the 
subject  is  seated.  Hold  the  sphygmometer  upon  the  distal  valve, 
and  empty  the  vein  above  by  pressing  a  finger  along  it  heart- 
ward,  and  determine  the  pressure  which  must  be 
exerted  to  prevent  the  vein  from  filling  from 
the  periphery.  This  will  just  balance  the  blood 
pressure. 

Pressure    in   the   capillaries.      Lay    the    subject's 

hand    palm    down    on    the    table,   rest    the   square 

C^2\         end    oi    the    little    glass    staff   upon    the    skin   at 

the   root   of    the   nail    of    the   middle   finger,    and 

press  upon   the    upper  end   of    the    staff   with  the 

ampulla  of   the  sphygmometer  until  the  skin  just 

blanches,   as    seen   through   the   end   of    the    staff, 

and  read  the  pressure.     The  end  of   the  staff  has 

an    area    of    half    a    centimetre    square    and    itself 

weighs    1    gramme.1      Add    this    to    the   pressure   indicated    by 

the    sphygmometer ;  the    sum    multiplied    by    four    gives    the 

capillary    pressure    per    square    centimetre. 

The  pressure  of  a  vertical  column  of  mercury  one  millimetre  long 
and  one  quarter  of  a  square  centimetre  in  section,  presses  with  a  force 
of  1-3598. 

Rate  of  blood  flow  in  the  capillaries.  Arrange  the 
web  of  a  frog  that  is  little  pigmented  under  the  microscope 
under  the  high  power,  and  adjust  a  chosen  length  of  capillary 
across  a  space    of  the  eye-piece  micrometer,  and  determine  its 

length. 


Fig.  49. 
Glass  Staff 
for  taking 
the  capillary 
pressure  with 
the  sphygmo- 
meter. 


]  A  column  of  mercury  760mm  lung  exerts  a  pressure  of  l-033  kilo  upon  the  square 
centimetre;  e.g.,  one  atmosphere. 


THE    FROG    HEART.  245 

Follow  the  movement  of  the  corpuscles,  and  practice  singling 
one  of  them  out  so  as  to  follow  its  course  from  one  end  of  the 
measured   length   of   capillary   to   the   other. 

Adjust  a  1/10  second  record  to  write  on  a  drum  going  at 
a  moderate  speed,  place  a  spring  key  in  circuit,  so  that  the 
style   vibrates   when    the    latter    is    closed. 

Depress  the  key  at  the  moment  a  corpuscle  enters  the 
measured  area,  and  release  the  key  the  moment  that  the 
passage  is  completed.  From  the  record  estimate  the  rate  per 
second. 

In  carrying  out  the  experiment  the  co-worker  should  take 
charge  of  the  writing  point,  turning  it  on  to  the  paper  when 
you  are  ready  and  off  again  each  time  a  record  is  completed. 

The   Prog-   heart. 

Enquired: — Frog-heart  recorder  with  parchment  paper  point  tipped 
with  a  glass  filament,  general  stand,  induct  orium  arranged  for 
interrupter  shocks,  light  electrodes,  2  keys,  and  7  wires. 

After  pithing  the  frog  plug  the   opening   to   arrest   bleeding 

Lay  the  frog  on  its  back  on  the  frog  plate,  divide  the  skin 
along  the  middle  line  for  the  length  of  the  sternum  without 
opening  the  abdominal  cavity.  The  sternum  is  then  cut 
through  transversely,  leaving  a  little  of  the  cartilage  below 
undisturbed,  and  is  then  completely  detached  laterally  and 
above,  care  being  taken  in  doing  so  to  avoid  injury  to  the 
parts  beneath. 

A  window  is  thus  made  over  the  heart  without  opening  the 
pericardium  or  the  abdominal  cavity. 

Next  open  the  pericardium,  raise  the  heart  with  the  glass 
seeker  and  find  the  frenum  on  its  dorsal  aspect ;  this  contains 
the  small  cardiac  vein,  divide  it  and  turn  up  the  heart.  Note 
the  sinus  forming  the  entrance  of  the  large  veins  into  the 
right    auricle. 


246 


CIRCULATION. 


Observe  that  contractions  follow  each  other  in  the  order  of 
sinus,  auricles,  ventricle,  and  bulb.  The  ventricle  becomes 
pale  and  rounded  at  the  systole,  each  section  of  the  heart 
diminishing    in   volume    as    it    empties    itself. 

Record  the  movements  by   GaskelVs   suspension  method. 


f 


O 


B 


=3       P 

c 


§ 


A 


-^ 


,y 


Jc 


A 


% 


H 


^ 


r^ 


Fifi.  50.  Frog-heart  recorder.  A  Aluminium  rod,  attached  to  the  hinge  H  and 
the  recording  arm.  The  heart  is  connected  to  it  by  the  hanging  thread.  S  Counter- 
poising spring  adjusted  by  the  slider  on  the  vertical  rod.  F  Wooden  base  clamped 
to  the  bracket  on  the  pillar  P  of  the  general  stand.  C  Loaded  cork  frog  plate. 
B  Side  bar  carrying  T  an  electric  style. 


A  fine  silk  thread  about  12cm  long,  moistened  with  normal 
saline,  has  a  single  loop  made  upon  it ;  this  is  slipped  over 
the  apex  and  is  tied  so  as  to  include  the  smallest  possible 
portion  of  the   tip. 

This  can  be  done  without  much  difficulty  if  the  ligature 
be  slowly  tightened  at  first  around  the  ventricle  until  it  slips 
to  the  exact  spot  at  which  it  is  to  be  fastened,  when  it  should 
be  drawn  tight  suddenly.  Secure  with  a  second  knot.  Then 
place  the  frog    with    the    frog    plate    upon    the    board    of    the 


STIMULATION    OF    THE    CRESCENT.  247 

recorder.  Attach  the  thread  by  slipping  it  between  the  end  of 
the  aluminium  and  the  recording  arm;  friction  will  hold  it  firmly. 

Adjust  the  counterpoising  spring  so  that  the  tension  may 
be  such  as  to  give  the  maximal  excursion  to  the  lever ;  see 
that  the  latter  is  horizontal.  Owing  to  the  oblique  position 
of  the  spring,  the  tension  remains  almost  constant  in  all 
positions  which  the  lever  will  assume.  The  heart  may  be 
stretched  to  a  considerable  extent.  Drum  at  slowest  speed, 
3  or  4  contractions  to  a  cm. 

Take  a  record  of  the  cardiac  movements.  The  contractions 
will  be  marked  by  down  strokes,  and  the  tracing  may  show 
sinus,  auricle,  and  ventricular  contractions.  The  first  is  usually 
absent,  and  the  distinctness  of  the  auricular  curve  is  much 
affected  by  the  pericardial  attachments,  which  vary  a  good 
deal  in  different  frogs.  It  is  necessary  to  look  for  and  divide 
any  restraining  tissue  if  the  movements  appear  hampered. 

Stimulation  of  the  crescent.  Find  at  the  sinus-auricular  junc- 
tion, the  curved  tendinous  line  of  demarcation  which  has  its 
convexity  turned  auriclewards.     This  is  known  as  the  crescent. 

Use  the  light  pair  of  electrodes  made  of  thin  copper  wires, 
mounted  on  a  cork  transfixed  by  two  pins  by  means  of 
which  they  are  to  be  fixed  to  the  frog  plate.  The  ends 
of  the  wires  are  to  be  placed  so  as  to  embrace  the  crescent, 
apply  interrupter  shocks  and  gradually  increase  their  strength. 
Fairly  strong  shocks  will  be  required.  The  heart  will  at 
first  beat  faster  for  a  few  beats  and  then  become  slowed, 
and  if  the  stimulus  be  .  of  sufficient  strength,  it  will  be 
arrested  in  diastole,  i.e.,  the  lever  will  rise  to  and  remain 
at  zero.  If  the  stimulation  be  continued,  the  heart  often 
begins    to    beat    again    (vagus    escape). 

The  first  few  quicker  beats  are  due  to  the  vagus  being 
a    vago-sympathetic    (Gaskell),    and    the    accelerator  fibres    are 


248  CIRCULATION. 

usually  called  into  action  first,  the  inhibitory  fibres  exhibiting 
some  delay  in  manifesting  their  action.  There  is  much 
variation  in  this ;  winter  frogs  show  it  most.  The  same 
result  obtains  if  the  trunk  of  the  vagus  or  its  cardiac  branch 
be    stimulated. 

The  performance  of  the  latter  operation  is  not  considered 
suitable  for  these  exercises,  as  the  branch  mentioned  is  very 
small  and  not  easy  of  access.  The  dissection  necessary  to  find 
this    nerve   is    given   in    the   appendix. 

Effect  of  atropine  and  muscarine  on  vagns  action. 
Next  having  surrounded  the  base  of  the  heart  with  small 
pieces  of  blotting  paper,  bathe  its  surface  around  the  crescent 
with  tincture  of  atrojoine  by  means  of  a  glass  rod.  Only 
apply  sufficient  to  wet  the  surface  itself.  In  a  few  minutes 
repeat  the  stimulation  at  the  crescent,  there  will  be  no 
response.     The   vagus    is    paralysed    at   its    termination. 

Muscarine.  Bathe  the  heart  as  before  with  a  dilute  solution 
of  muscarine.  In  a  little  time  the  heart  will  come  to  a  stand- 
still in  diastole.  This  resembles  forcible  action  of  the  vagus. 
If  too  much  atropine  has  been  used,  the  effect  will  not 
manifest    itself. 

Again  bathe  the  heart  with  atropine,  the  heart  will  presently 
begin  to  beat  again ;  this  is  taken  to  prove  that  the  muscarine 
acted   as   a   stimulant   to   the  vagus. 

Effect  of  heat  and  cold  on  the  rate  of  the  heart's 
contraction. 

Cool  some  normal  saline  to   5°  C.  by  means  of  ice ;  with   a 

pipette  flow  it  drop  by  drop  on  to  the  heart  whilst  the  latter 

is    recording  its  movements.      The   rate    will    be    slowed.      Let 

the   heart  recover  its  previous  rate,   then  drop  upon   it  water 

which  has  been   heated  to   35°  C.     The  rate   will  be  increased. 


STANNIUs'    LIGATURE.  249 

Stannius'  ligature.1  No.  1.  Arrange  the  pithed  frog  to  24 
record  by  the  suspension  method,  and  pass  a  ligature  under 
the  aortse,  bring  it  round  to  the  dorsal  side  of  the  heart, 
and  tie  a  loop ;  apply  this  to  the  line  of  junction  of  auricles 
and  sinus,  and  ascertain  that  it  is  placed  in  contact  with 
the  crescent,  then  draw  it  tight.  The  ventricle  will  stop 
beating.  This  arrest  always  lasts  for  a  considerable  time, 
and  may  be  permanent.  Failure  is  usually  due  to  the  ligature 
not  having  been  placed   upon   the  crescent. 

Affix  the  light  electrodes  as  for  crescent  stimulation  only  in 
this  case,  against  the  ventricle  itself,  and  arrange  to  stimulate 
with  single  shocks.  With  a  sufficient  stimulus,  the  heart  will 
respond  with  a  single  contraction   at   each   stimulus. 

Latent  period.  Place  the  drum  contact  in  P  circuit, 
drum  at  a  moderately  fast  speed.  Inscribe  a  1/10  second 
time  tracing  on  the  paper.  Bring  the  recording  point  into 
contact  with  the  paper  for  one  revolution  of  the  drum  only 
at  a  time.  Mark  the  point  at  which  the  stimulus  was  thrown 
in.  Measure  the  duration  of  the  period,  and  compare  it  with 
that  already   obtained   with   striped   muscle. 

Cardiac  tetanus.  After  concluding  the  last  experiment, 
attempt  to  induce  tetanus  by  stimulating  the  ventricle  with 
interrupter  shocks.  Cut  out  the  drum  contact  and  connect 
the   interrupter   in   the   P  circuit. 

It  will  be  found  that  the  most  rapid  succession  of  stimuli 
will  only  yield  an  irregularly  continued  contraction,  but  so 
far  incomplete  that  individual  twiches  are  still  represented 
upon   the   curve. 

1  Stannius'  ligature.    No.  2  is  applied  in  a  similar  manner  to  the  line  of  junction 
of  the  auricles  with  the  ventricle.     It  is  followed  by  a  revival  of  the  ventricular  beats 
the  auricles,  however,  remain  at  rest. 


250  CIRCULATION. 

Effect  of  extract  of  Suprarenal  Capsule  on  the  flow 
through  the  blood  vessels. 

Required : — Glass  aortic  canula  for  the  frog,  connected  by  a  rubber 
tube  15 cm  long  to  a  glass  funnel;  a  50 cc  measure  with  a  funnel  and  a 
retort  stand. 

Pith  the  frog  and  plug  the  opening. 

After  exposing  the  heart  through  a  window  in  the  sternum, 
introduce  the  canula  into  the  left  aorta  and  tie  it  in.  Ligature 
the  right  hand  one. 

Incise  the  sinus  so  that  the  fluid  may  pass  freely  from 
the   A'eins. 

The  frog  is  to  be  suspended  by  its  head  with  threads  attached 
to  the  ring  of  a  retort  stand,  and  with  its  legs  hanging  into  the 
funnel  in  the  mouth  of  the  glass  measure. 

The  funnel,  with  rubber  tube  attached  and  closed  with  a  clip, 
is  supported  on  another  ring  of  the  retort  stand  on  a  level 
with  the  frog's  head.  Fill  it  and  the  canula  with  normal  saline 
and  connect  them,  being  careful  to  exclude  air  bubbles. 

The  saline  solution  is  next  caused  to  flow  through  the  vascular 
system  of,  the  frog,  and  the  quantity  which  passes  collects  in 
the  measure. 

The  height  of  the  fluid  is  read  off  at  short  intervals  (1  to  3 
minutes),  and  is  recorded  in  a  tabular  form  in  your  note-book. 

After  a  constant  rate  of  flow  is  established,  in  from  5  to  10 
minutes,  substitute  suprarenal  extract  for  the  saline. 

I)«'tach  the  rubber  tube  from  the  canula,  empty,  and  fill  the 
funnel  and  tube  with  the  extract;  connect  again  to  the  canula 
and  continue  the  observation. 

A  decrease  will  indicate  obstruction  to  the  flow  due  (Oliver 
and   Schafer)  to  contraction  of  the  small  arteries. 


CHAPTER   XXXV. 

VISION. 

Field  of  vision.     Perimeter.     The  meridional  rays  represent 
an  hemispherical  surface  in  space. 


26 


FlG.  51.     Perimeter. 


252  vision. 

They  are  divided  into  degrees,  and  each  carries  a.  curser 
C  bearing  a  piece   of    white  or  coloured  paper. 

The  operation  is  to  be  conducted  with  movable  lights  in  a 
darkened  room. 

The  subject  throughout  the  observation  looks  directly  with 
one  eye  at  the  point  of  fixation  F,  which  is  a  mirror  with 
•cross  lines.  He  knows  that  his  eye  is  centred  when  the 
cross  is  centred  in  the  reflection  of  his  pupil. 

His  chin  must  rest  on   R   (suitably  adjusted   for  height). 

The  cursors  are  moved  in  succession  from  the  equator 
towards    F   by  the  observer  until  just  visible   to  the   subject. 

Determine  successive!}^  the  outlines  of  the  fields  of  the  two 
eyes  for  white,  red,  green,  and  blue  and  transfer  the  results 
to  a  printed  chart. 

Remember  that  the  point  of  fixation  corresjDonds  to  the 
fovea  centralis  of  the  retina.  The  retinal  image  of  the  field 
is  reversed  in  all  directions. 

Compare  the  fields  of  the  two  eyes. 

Examination  of  the  interior  of  the  eye.     The   ophthal- 
moscope. 

Light  entering  the  eye  is  reflected  from  the  retina  in  the  same 
direction  as  that  from  which  it  entered.  You  cannot,  therefore, 
see  into  another  person's  eye  without  employing  some  artifice, 
because   your  head  intercepts   the   light. 

Artificial  eye: — A  round  (pill)  box  of  about  22 mm  depth, 
blackened  internally,  in  the  lid  of  which  is  a  lens  with 
a  principal  focus  of  the  same  length  as  the  depth  of  the  box. 
The  bottom  of  the  box  represents  the  retina ;  it  has  some 
printed  matter  upon  it,  and  three  small  apertures  through  it, 
placed  l-5nim  from  each  other.  The  latter  can  be  blocked 
at  will  with  a  piece  of  black  card,  so  that  the  apertures 
appear    as    black    dots   when   viewed    from    the    front. 


OPHTHALMOSCOPE. 


253 


Set  up  the  model  in  a  darkened  room  with  the  lens  towards 
you    and    on    a   level   with  your    eye. 

Direct  method  of  using  the  ophthcdmoscopc.  First,  with  the 
apertures  closed,  endeavour  to  look  into  the  eye  through  the 
lens,  moving  your  eye  and  a  light  in  all  directions  to  do  so. 
You  will   not  succeed. 


32Ctn  | 


▼    ■ 


FIG.  52.  E  Eye  model.  L  Light  opposite  the  apertures  in  the  fundus ;  the 
occluding  card  is  not  shown.  *  Lateral  position  of  the  light  when  the  mirror  M  is 
in  use.  A  Amplifying  lens.  R  Is  placed  in  line  with  the  retina  of  the  observer's 
eye.  O  Object,  F  and  R  (observer's  retinal)  real  images.  C  Cerebral  inversion  of 
the  last.     M   Position  of  the  mirror ;  the  central  aperture  of  which  is  exaggerated.. 


Secondly  place  the  light  behind  the  model,  open  the  aper- 
tures, and  on  looking  into  it  you  will  see  them  and  their 
edges  distinctly,  the  apertures  being  now  radiant  points  of 
light.  Vision  will  be  most  distinct  at  a  short  distance  from 
the  model.  This  corresponds  to  the  direct  method  of  using 
the   ophthalmoscope. 

Now  close  the  apertures,  place  the  light  on  one  side  of 
the  model  and  hold  the  mirror  of  the  instrument  in  front  of 
and  close  to  your  own  eye,  look  through  its  central  aperture 
and  at  a  distance  of  about  7 cm  direct  light  into  the  model. 
The  fundus  and  the  points  previously  visible  because  they 
emitted  light  are  now  seen  because  they  reflect  light  in  the 
required   direction.      The    mirror  enables    you    to   get    over    the 


254  vision. 

difficulty  produced  by  the  interposition  of  your  head  in 
the   path    of   entering   rays.     Is   the  image   erect   or  inverted  ? 

Again  illuminate  the  eye  from  behind,  with  the  apertures 
open,  and  hold  a  white  screen  in  front  of  the  model,  so 
that  the  emergent  rays  shall  fall  uj)oii  it.  Note  that  the 
light  does  not  come  to  a  focus  at  whatever  distance  the  screen 
be  held.  The  rays  practically  emerge  parallel.  In  consequence 
of  this  the  observer's  eye  should  have  normal  vision  (be 
emmetropic)  and  be  in  a  state  of  negative  accommodation,  in 
order  that  it  may  be  able  to  focus  the  parallel  rays  on  to  its 
own  retina. 

Indirect  method  of  examination   (Fig.    52). 

Next  mount  a  lens  of  12  to  14  D.  focus,  and  the  mirror 
in  separate  cork  holders.  Level  them  with  the  aufcincial  eye. 
Throw  the  light  through  the  back  of  the  model.  Place  the 
lens  4.5  to  5cm  in  front  of  the  latter  and  look  at  its  interior. 
The  apertures  will  be  seen  through  the  lens,  but  you  will 
have  to  place  yourself  some  distance  away  (40  to  50 cm)  from 
the   model   in   order   to   see   them   distinctly. 

Then  follow  the  behaviour  of  the  liarht  with  the  screen. 
A  sharp  image  (inverted)  of  the  apertures  in  the  fundus  will 
be  formed  at  some  8  to  10cm  from  the  lens.  The  rays  which 
form  this  image,  on  being  traced  beyond  it,  diverge  into 
expanding  cones,  and  these  gaining  access  to  your  eye  are 
brought  to  a  focus  upon  your  retina,  and  there  form  another 
image  (erect).  The  latter  is,  however,  interpreted  as  an 
inverted  image  by  the  brain,  and  appears  projected  at  about 
30 cm   (12   inches)    from   the   observer. 

Close  fche  apertures  in  the  fundus,  place  the  light  on  one 
side  of  the  model,  and  adjust  the  mirror  so  as  to  illuminate 
.the  fundus;  the  black  spots  and  the  type  will  be  seen  inverted 
and   enlarged. 


SCHEINER  S    EXPERIMENT. 


255 


The  lens  should  be  moved  to  and  from  the  eye  until  the 
edge  of  the  pupil  falls  beyond  its  margin;  it  should  also  be 
slightly  inclined  from  the  perpendicular  in  order  to  prevent 
the   disturbance   caused   by   reflections   from   its   surface. 

The  two  methods  should  be  practised  upon  the  eye  of 
another  person. 

Accommodation.  Scheiner's  experiment.  Prick  two  holes 
in  a  card  near  each  other,  so  that  they  fall  within  the  diameter 
of  the  pupil,  hold  the  card  close  to  the  eye  with  the  holes 
placed  horizontally  and  look  through  them  simultaneously  at 
two  pins  stuck  vertically  into  corks  (which  rest  upon  a  table), 
the  one  at  60 cm  and  the  other  at  25 cm  from  the  eye  in  the 
same  line  of   vision. 


Fig.  53.  N  and  F  the  near  and  far  pins.  D  the  card  with  its  two  holes  in  each 
case.  R  the  blocked  aperture.  C  reversal  by  the  brain.  The  course  of  the  rays  of 
light  from  the  non-focussed  object  are  represented  by  dotted  lines. 


First  focus  the  distant  pin,  three  images  will  appear.  The 
central  one  is  the  single  sharp  image  of  the  further  pin. 

Prove  that  the  two  side  ones  are  separate  images  of  the  near 
pin,   which  are  insufficiently   converged  to  fall    upon   the  same 


256 


VISION. 


part  of  the  retina,  as  follows: — On  blocking  the  right  hand  hole 
in  the  card,  the  left  hand  image  will  disappear. 

The  reversal  by  the  brain  is  to  be  taken  into  account  in 
determining  upon  which  side  of  the  retina  the  disappearance 
occurs,  e.g.,  the  side  to  which  the  image  is  referred  in  the  field 
of  vision. 

Repeat  the  experiment,  but  on  this  occasion  focus  the  near 
image.  Three  images  will  again  appear,  but  on  blocking  the 
right  hole  in  the  card  the  image  on  that  side  disappears.  This 
proves  that  the  eye  is  over-accommodated  for  the  distant  pin, 
and  that  in  consequence  the  rays  must  have  crossed  in  front 
of  the  retina. 

28  Listing's   diagrammatic   eye.1     Make   use   of    the   rule   of    three 

formula   given   in   the  figure    (Fig.  54)   in   the   following   calculations : — 


A:l5mw  ::B:  x 

Fig.  54. 

Marriott's  experiment  and  measurement  of  the  blind  spot. 
Seat  yourself  at  arm's  length  in  front  of.  a  sheet  of  paper 
pinned    to    a    wall    (drawing    board).       Mark    a    spot    straight 


'Accommodation  and  refraction  of  the  eye.  (Donders.  New  Sydenham  Society, 
'  For  the  ordinary  eye  we  substitute  one  with  a  cornea,  whose  radius  of 
curvature  is  only  .",111111,  while  behind  this  is  merely  vitreous  or  aqueous  humour, 
without  crystalline  lens,  and  with  a  length  of  visual  axis  of  20mm.  In  such  an 
eye  retinal  images  would  have  the  same  magnitude,  the  same  distinctness,  and 
the  Bame  position  which  they  exhibit  in  the  emmetropic  eye,  with  its  cornea  of 
nearly  8 nun  radius  of  curvature,  its  crystalline  lens  of  a  little  more  than  4:3  nun, 
focus  distance,  and  its  visual  axis  of  a  little  more  than  22mm,  and  it  can,  there- 
fore,  really   be  substituted   for  this  last." 


RETINAL    STIMULATION.  257 

before  you  and  look  at  it  fixedly  with  one  eye  (the  dis- 
engaged eye  being  closed),  then  carry  the  point  of  a  pencil 
outwards  from  the  marked  spot  until  it  passes  out  of  view ; 
mark    where    this    occurs. 

Define  the  blind  area  in  all  directions ;  the  beginning  of 
the    large   vessels    may   be   also   noted. 

Measure  the  diameter  of  the  outline  thus  obtained,  and 
the  distance  of  the  paper  from  the  eye,  and  calculate  the  size 
of   the  retinal  image  from  the  reduced  eye. 

Discriminative  power  of  the  eye  for  detail.  Place 
a  card  on  which  parallel  lines  1 mm  thick  are  ruled  at  in- 
tervals of  1 mm  from  each  other  upon  a  well  illuminated  wall, 
and  measure  the  greatest  distance  at  which  you  are  able  to 
recognise  the  lines  distinctly  from  each  other.  By  means  of 
the  reduced  eye,  determine  their  distance  from  each  other 
in  the  retinal  image.  Compare  the  ascertained  intervals 
with  the  distance  which  separates  the  outer  segments  of  the 
cones  from  each  other   in   the  fovea  centralis.      (4/a). 

Periodic  stimulation  of  the  retina  with  "white  light. 
The  experiments  are  performed  with  discs  of  cardboard  divided 
into  differently  proportioned  white  and  black  sectors,  &c. 
The  collaborator  rotates  the  disc,  whilst  the  observer  stations 
himself  facing,  and  at  a  distance  of  about  2*5  metres  for  a 
disc  of  20 cm  diameter.  Or  the  observer  looks  at  the  reflection 
of  the  disc  in  a  mirror  at  half  the  distance.  He  can  then 
operate   the   discs   himself. 

1.  White  and  black  hemidiscs.  Rotate  at  a  gradually 
increasing  speed ;  just  before  the  sensation  becomes  fused  into 
a   silver   grey   there  is  a   marked   period  of  flicker. 

2.  A  sixth  sector  white.  Rotate  slowly  at  rather  more 
than    one    turn    a    second ;    follow    the    retreating   edge   of   the 


258  vision. 

black;  a  slight  radial  shadow  will  be  seen  within  the  white 
(Charpentier).  Under  very  favourable  conditions  a  second  and 
even  a  third  may  be  observed. 

This  effect  is  ascribed  to  the  oscillatory  response  of  the 
retina  to  the   stimulus. 

3.  The  spectrum  top  of  Bentham.  The  white  hemidisc  is 
•divided  into  four  equal  sectors ;  in  each  one  of  these  a  set 
of  concentric  lines  is  described,  in  the  first  sector  near  the 
periphery,  in  the  second  a  quarter  diameter  nearer  the  centre, 
and   so   on  for  the  remaining  sectors. 

Observe  the  colour  effects  produced  on  rotating  the  disc  at 
a  medium  speed  ;  the  effect  is  reversed  when  the  direction  of 
rotation  is  changed. 

The  illumination  should  not  be  too  brilliant.  A  slightly 
yellow  light  is  frequently  more  efficient  than  white  light  in 
producing  the  effect. 


itln i.i  ilia 


CHAPTER   XXXVI. 

THE   CUTANEOUS    SENSES. 

The  subject  keeps  his  eyes  closed  and  indicates 
to  the  observer  the  nature  and  intensity  of  the 
sensation  produced.  It  is  sufficient  in  the  following- 
exercises  to  detect  a  marked  difference  in  an}^  one 
sensation ;  the  exact  evaluation  of  the  difference 
need  not  be  attempted. 

Tactile  and  pressure  senses.  Explore  the  lips, 
skin  of  the  face,  dorsum  of  the  hand  and  fore- 
arm   with   von    Frey's  hair   Eesthesiometer. 

For  the  least  perceptible  stimulus  the  human 
hair,  and  for  less  sensitive  parts  the  horse  hair, 
instrument   is   used. 

A  given  length  of  each  hair  is  capable  of 
exerting  a  certain  maximal  pressure,  which  can  be 
evaluated  in  grammes  per  square  centimeter.  For 
convenience  the  hair  is  placed  within  a  sheath, 
by  means  of  which  the  exposed  length  of  hair 
can  be  varied,  and  with  it  the  pressure  which 
can   be   exerted. 

The  after  sensation  is  frequently  very  marked, 
the  sensation  lasting  some  time  after  the  stimulus 
is    removed. 

Minimal  stimuli  applied  to  the  face  produces 
tickling    instead    of    a    markedly   tactile    impression. 

The  tactile  sensation  passes  into  a  feeling  of 
pressure  with  an  increase  of  the  stimulus.  The 
horse    hair    may    even    induce    pain. 


<l  1 1  M  I  I  I  I  i 

n 


V 


H 


Fig.  55. 

iEsthesio- 
meter. 

H  Human 
or  horsehair 

S  Movable 
sheath  by 
means  of 
which  the 
length  of 
hair  expos- 
ed may  be 
varied. 


29 


260 


THE    CUTANEOUS    SENSES. 


With  a  pair  of  dividers  find  the  least  distance  at  which 
the  points  can  be  distinguished  as  two  separate  impressions 
when  they  are  applied  simultaneously  to  the  skin.  Measure 
the   intervals  with   a   millimetre   scale. 

Test  the  tip  of  the  tongue,  the  skin  of  the  lips,  forehead, 
cheeks,  hand,  forearm,  and  back   of   the  neck. 

The  pain  sense.  Explore  the  skin  of  the  hand  and  forearm 
with  Aly's  instrument,  which  consists  of  a  long  needle  kept 
projecting  from  a  sheath  by  means  of  a  light  spring.  The 
pressure    which    is    required    to    produce    a    feeling    of   pain    is 

read  off  in  grammes  upon  the  gradua- 
tions on  the  sheath.  The  camel  hair 
brush  at  the  other  end  of  the  instru- 
ment is  the  common  clinical  means 
used  to  detect  diminution  of  sen- 
sibility. 

By  arming  the  end  of  the  needle 
with  a  small  piece  of  cork  it  can  be 
used  for  exploring  the  pressure  sense. 

Use  the  von  Frey  human  hair  for 
exploring  the  conjunctiva  for  the 
pain   sense. 

Hot  and  cold  sense.  Use  Gold- 
scheider's  metal  rods  (lcm  thick  by 
9  long).  Explore  the  back  of  the 
hand  and  forearm  by  touching  the 
surface  lightly  with  the  tip  of  the  rod. 

First  explore  for  spots,  which  at 
once  respond  by   a  feeling  of  cold. 

The  metal  is  usually  cold  enough 
at  ordinary  temperatures,  and  need 
not  therefore  be  specially  cooled. 


n        m 
Aly's  Ms\  besiometer. 


HOT    AND    COLD    SENSE.  261 

With  a  rod  heated  in  water  at  70°  C.  explore  for  the 
hot   spots. 

In  both  cases  mark  the  spots  where  the  two  senses  are 
most  acute,  and  those  where  a  definite  absence  of  both  can 
be  detected. 


APPENDIX    TO    THE    EXPERIMENTAL    SECTION. 

Dissection    for    exposing    the    Vagus    nerve    in     fche    frog". 

The  pithed  frog  is  laid  upon  its  back  on  the  frog  plate,  is  freely  incised 
mesially  through  skin  and  then  sternum. 

The  edges  of  these  are  drawn  widely  apart  and  kept  so  with  threads 
pinned  to  the  plate.  Restraining  connective  tissue  is  divided  close 
to  the  bone,  and  the  attachments  of  the  pericardium  are  carefully  re- 
moved around  the  heart. 

A  glass  tube,  1*5  to  2cm  wide  according  to  the  size  of  the  frog,  is 
passed  down  the  oesophagus  as  far  as  it  will  go  ;  this  stretches  the 
neighbouring  structures. 

From  the  angle  of  the  jaw  a  somewhat  deeply  situated  and  thin 
muscular  band,  composed  of  the  petrohyal  muscles,  extends  to  the  region 
of  the  heart  (strictly  speaking,  to  the  hyoid  cartilage). 

The  Vagus,  dividing  into  its  cardiac  and  laryngeal  branches,  lies 
beneath  the  lower  edge  of  this  muscle,  and  must  be  carefully  sought  for, 
as  it  is  usually  very  small. 

The  petrohyals  lie  above  the  pronounced  levator  anguli  scapulas 
muscle  that  slants  down  and  outwards  to  the  upper  limb. 

The  petrohyals  are  crossed  by  two  distinct  nerves. 

One,  the  glossopharyngeal,  sweeping  in  a  curve  from  the  angle  of  the 
jaw,  passes  upwards  to  disappear  amongst  the  muscles  of  the  floor  of 
the  mouth. 

The  other,  the  hypoglossal,  usually  piercing  the  levator  anguli  scapulas 
curves  inwards  in  the  same  direction  as  the  first,  to  disappear  nearer 
the  middle  line. 

The  Vagus  must  be  carefully  separated  from  the  muscle  for  as  long 
a  distance  as  possible.  A  moistened  thread  is  passed  beneath  it  and  is 
tied  near  the  angle  of  the  jaw.  The  nerve  is  cut  between  the  jaw 
and  the  ligature,  and  it  can  then  be  raised  clear  of  its  surroundings  for 
the  application  of  the  electrodes. 

Du  Bois  Reymond's  compensation  method  for  the  measure- 
ment of  the  electromotive  force  in  muscle  and  nerve.  Establish 
the  connections  as  shown  in  the  figure,  with  one  gap  of  the  metre  bridge1 

'Instead  of  the  metre  bridge,  the  zig-zag  resistance,  fig.  46,  pg.  234,  may  be  employed, 
as  its  wire  is  longer  and  thinner,  and  smaller  differences  can  be  more  readily  detected 
by  its  means.  This  wire  is  of  about  20  ohms  resistance  and  is  divided  into  10  parts, 
fractions  of  which  can  be  measured  with  a  foot  rule.  The  portions  in  contact  with 
the  pulleys  around  which  the  wire  is  stretched  must  be  omitted  from  the  length 
measured. 


APPENDIX    TO    EXPERIMENTAL    SECTION. 


263 


open. 


Place 


rider    against 


Fig.  57.  E  non-polar- 
i sable  electrodes.  C 
commutators.  G  gal- 
vanometer. D  Daniell 
cell.  B  gap  in  bridge 
bar.     J  rider,     a  and  b 

on  the  bridge  wire,  the  length  of  which  is 

1000  mm. 


the  bridge  wire  a  few  cms  from 
its  extremity  a  and  turn  the 
commutator  into  the  position  in 
which  the  G  deflection  is  smallest, 
then  move  the  rider  J  until  a 
balance  is  obtained,  e.g.,  the  spot 
of  light  on  the  G  scale  stands  at 
Zero.  This  will  be  very  near  the 
left  end.  This  fraction  a  of  the 
wire  compared  with  its  total 
length  gives  the  E.M.F.  of  the 
tissue,  &c. ,  in  terms  of  a  Daniell 
(1-1  volt). 


As  1000  is  to  aJ  so  is  1*1  (volts)  to  x  =  volts  of  the  tissue. 

Measurement  of  resistance  with  the  bridge  box.  When 
C  and  d  are  in  the  same  proportion  as  a  and  b  there  is  no  deflection 
of  the  galvanometer.  This  condition  is  sought  for  and  is  known  as 
the  zero  method. 


»     a:v::c:d 


Fig.  58.  Diagram  of  Paul's  resistance  box  and  Wheatstone's  bridge.  P  Shows 
the  connections  of  the  terminals  in  the  actual  box  and  W  the  same  transferred  to 
the  usual  diagram  of  Wheatstone's  bridge.  B  Battery.  G  Galvanometer.  L  Line 
or  the  points  to  which  the  unknown  resistance  X  is  to  be  connected.  B  Spring 
key  which  on  closure  leads  to  the  proportional  arms,  r  to  R  the  variable  resistance 
R  arranged  in  four  rows  ;  the  movable  plugs  p  are  connected  for  zero  or  no 
resistance  in  diagram  P. 


2U 


APPENDIX    TO    EXPERIMENTAL    SECTION. 


Measurements  are  made  as  follows  : — c  being  found  by  trial. 
As      a     is  to       b     so  is     c     to      d     ohms. 


„     10     , 

10   „ 

»     10     , 

100   ,, 

„      10     , 

1000   „ 

„ioo     , 

10   ,, 

„  1000   , 

10   ,, 

3 

„   3 

3 

„  30 

3 

„  300 

3 

„   0-3 

3 

„   0-03 

Fig.  59.     Paul's  Bridge  Box. 

Kohlra usche's    method   fop    measuring    the    resistance    of 
electrolytes  by  means  of  alternating  currents  and  telephone. 


£ 


/* 


SL.IDE.  METRE  BRIDGE! 


JX_ 


JOOO 


T 


IB  L.  B  PH  ON  &  • 

Fig.  60.     Kohlrausche's  Method. 

Insert  a  variable  resistance  R  (of  bridge  box)  into  one  gap  of  the  metre  bridge, 
and  the  unknown  resistance  into  another  corresponding  one  at  the  other  end. 
Connect  the  telephone  to  opposite  ends  of  the  bridge  wire,  and  the  wires  from  the 
S  coil  of  an  inductorium  to  the  centre  of  the  copper  bars  on  one  hand  and  the 
rider  J  on  the  other.  Or  the  bridge  box  may  be  employed  instead  by  substituting 
SC  for  the  battery  B,  and  the  telephone  for  the  galvanometer  Q. 


MEASUREMENT    OF    RESISTANCES. 


265 


Set  J  at  500,  hold  the  telephone  to  your  ear  (two  telephones  are 
better),  and  adjust  R  until  the  sound  fades  to  the  lowest  the  resist- 
ance will  permit,  e.g.,  find  the  two  resistances  between  which  there  is 
silence  ;  then  move  J  until  two  points  are  again  found  between  which 
silence  occurs. 

Then,  as  the  lengths  of  wire  on  each  side  of  J  are  to  each  other, 
so  is  R  to  X. 

The  inductorium  must  be  placed  in  an  adjacent  room  so  that  it  be 
not  directly  audible,  and  the  S  coil  must  be  adjusted  to  give  a  well 
marked  sound  in  the  telephone.  If  two  telephones  are  employed,  one 
to   each   ear,  a  collaborator  will  be  required  to  adjust  the  resistances. 

Measurement  of  resistance  of  a  galvanometer  op  cell  by  the 
half  deflection  method. 


GALVANOMETER  BATTERY 

FIG.    61. 


(a)  Galvanometer:  —  Battery  (1  Daniell)  short-circuited  on  closure  of 
key  through  the  shunt  wire  S.  R  variable  resistance.  G  the  galvano- 
meter. Vary  S  (No.  18  wire,  25  to  30  cm  long,  with  G  about  6000  ohms) 
until  the  deflection  of  the  galvanometer  is  about  20  cm  on  the  scale. 

Then  increase  R  until  the  deflection  is  reduced  to  one-half.  The  added 
resistance  R  will  equal  the  resistance  of  the  galvanometer. 

(I>)  Battery : — Transfer  the  shunt  wire  S  to  the  terminals  of  the  galvano- 
meter (No.  14  wire,  a  straight  piece)  and  adjust  to  the  same  deflection  as 
before.  Increase  R  until  half  the  deflection  is  obtained.  R  will  represent 
the  resistance  of  the  battery. 

It  is  best  to  take  an  odd  number  of  batteries,  5  or  7  coupled  end 
on,  with  an  even  number  coupled  in  opposition — zinc  to  zinc,  so  that 
there  will  be  E.M.F.  only  from  one  cell  in  the  G  circuit. 

The  total  resistance  divided  by  the  number  of  cells  gives  the  average 
resistance  for  one. 


266 


APPENDIX    TO    EXPERIMENTAL    SECTION. 


Power  distribution  for  actuating  recording  cylinders  in  the  Experi- 
mental Laboratory,  Physiological  Department,  Yorkshire  College. 

A  water  motor  (Chicago  stop)  is  geared  by  means  of  a  cord  M  to  a 
.54»inch  bic}-cle  wheel  W.  G  is  a  pressure  gauge  on  the  motor  side  of 
the  tap  T,  which  leads  by  a  short   branch   from  a   l|-inch  main   direct 


Fl«.  62. 


POWER    DISTRIBUTION    IN    THE    LABORATORY.  '267 

from  the  supply  in  the  street.  A  adjustable  pulley  for  regulating  tin- 
tension  of  the  cord.  The  wheel  is  set  to  run  at  one  revolution  a 
second  for  ordinary  class  purposes,  and  is  geared  to  the  shaft  S  by 
means  of  a  cord  which  connects  the  reducing  pulley  R  to  the  driving 
pulley  P.  The  cord  is  guided  in  the  required  change  of  direction  by  a 
system  of  guide  pulleys  D,  shown  in  side  view  in  the  right-hand  figure. 
By  means  of  these  the  cord  is  also  taken  to  the  counterpoise  C,  which 
maintains  an  equable  tension.  Cord  r  to  cone  R  on  the  wheel ;  W 
marks  the  position  of  the  latter  in  section. 

The  shaft  S  is  of  1-inch  steel  tubing,  carried  on  Bown's  ball 
bearings  at  intervals  of  30  inches.  Upon  it  speed  cones  P'  are 
threaded,  one  for  each  recording  drum  in  the  laboratory. 

Each  speed  cone  is  15  inches  in  diameter,  and  is  built  up  of 
mahogany.  It  turns  once  in  1^  seconds  when  W  is  making  one  turn  a 
second.  The  cord  connection  to  a  drum  is  shown  in  Fig.  34  I,  where 
the  tension  pulley  L  and  guide  K,  immediately  over  each  work  table, 
are  also  seen. 

As  all  the  rolling  parts  are  either  borne  upon  centres  U  (a  pulley 
shown  in  section),  or  in  ball  bearings,  friction  is  reduced  to  a  minimum; 
and  as,  furthermore,  the  wheel  is  loaded  with  lead  L,  and  the  cylinder 
cones  on  the  tables  are  heavy  (Fig.  34  D),  these  together  form  a  system 
of  fly  wheels  which  produce  great  steadiness  of  running. 

With  a  water  pressure  of  25  pounds  at  the  motor,  a  very  constant 
rate  of  movement  is  obtained,  which  is  not  appreciably  affected  by 
starting   and   stopping   the  drums. 

Under  a  fortieth  of  a  horse  power  is  required  to  drive  the  whole 
apparatus   in   the   laboratory. 

The  speed  of  W  is  ascertained  by  counting  its  revolutions  by  a 
watch,  for  which  purpose  a  white  mark  X  serves  as  a  guide. 

The  wheel  is  fixed  to  the  wall  by  a  three-branched  bracket,  seen 
behind  R,  and  is  mounted  upon  it  in  the  same  way  as  the  pedal  of 
a   bicycle  on  its  crank. 

Simplicity  and  economy  in  working  are  salient  features  of  this 
arrangement. 


INDEX. 

PAGE 

Acetone    

.  128 

,    158 

Blood  flow        

Adenoid  tissue 

.     36 

,,     Hayem's  fluid       

After-hardening 

.     18 

,,     plasma    

,,     load,  muscle  .. 

.  220 

, ,     pressure 

Albumin,  acid 

.  134 

,,     serum      

,,         alkali 

.  133 

,,     spectra 

egg          ...     . 

.  128 

,,     sugar  in 

, ,         serum 

.  145 

Bone,  dry 

,,         estimation 

.  17C 

1,  145 

,,      growing  head       

Albuminuria    

.  170 

,,      shaft       

Albumoses        

.  134 

,,      softened        

Albumosuria    

.  171 

Bread        

Amalgamation,  zinc 

.  184 

Bronchus *... 

Amoeboid  motion    ... 

.     59 

Brownian  motion 

Aorta         

.     56 

Apparatus  for  Histology 

.       4 

Capillary  electrometer 

,,              Chemistry    .. 

.  119 

Carbohydrates         

,,              Experimental 

181 

,,              table  of  tests 

Artery  and  Vein 

.     55 

Carbol-iron  test       

,,             ,,        distended.. 

.     55 

Cardiac  tetanus       

Auerbach's  plexus 

.     73 

Cardiogram      

Cartilage,  elastic     

Bacteria 

.     12 

,,         hyaline 

Balsam,  mounting 

.     13 

,,         white-fibro    

Barfoed's  test 

122 

Caseinogen       

Bichromate  cell      

.  185 

Cerebellum       

Bile    

.  155 

Chronogram     

Blood         

.  144 

Circulation,  frog     

,,     amphibian  and  human 

57 

Cochlea     

,,     cells,  red        

.     58 

Commutator    

,,        ,,      counting       

.     30 

Connective  tissue 

,,        ,,      white    

.     59 

Conus  medullaris    

,,     coagulation    

.  146 

Cotton  fibre     

,,     films        

.     60 

Cornea       

270 


INDEX. 


Corneo-sclerotic  junction 
Corpora  quadrigemina  .. 
Cover-glass,  application 
, ,  cleaning 

Cream        

Creatinin 

Crura  cerebri  ... 
Curara  experiment 
Cutaneous  senses 
Cystin       

Daniell  cell 
Decalcification 
Demarcation  current 
Description  of  object 

Dextrin     

Dextrose 

Digestion,  acids  in . . . 

,,  gastric   ... 

, ,          pancreatic 
Drawing 

Elastic  tissue 

Electric  style 

Electrodes,  hand    ... 

,,  non-polarisable 

Electrotonus    

Embedding,  celloidin 
gum    ... 
, ,  paraffin 

Emulsification 

Endothelium    

Epithelium,  ciliated 

, ,  chloroform 

,,  isolated 

, ,  columnar 

Fallopian  tubes 

Fat     

,,    in  milk       

Fatty  tissue  

Fehling's  test 


PAGE 

102 

96 

11 

7 

137 

167 

96 

223 

259 

172 

184 

19 

236 

11 

125 

121 

142 

140 

143 

8 

35 

209 

187 

232 

231 

26 

25 

20 

127 

28 

31 

32 

31 

30 

100 
127 
137 
36 
122 


PAGE 

Fermentation  test 175 

Fibrin,  structure    61 

Flesh         138 

Flour,  wheat 139 

Frog,  dissection,  dorsal         ...  194 

,,              ,,           ventral      ...  16 

,,       heart      245 

,,          ,,      current       237 

Galvani's  experiment     198 

Galvanic  cell    183 

Ganglion,  dorsal  root     92 

,,         sympathetic 92 

General  stand 209 

Glands,  pancreas    77 

,,        parotid      76 

,,        submaxillary    77 

,,        injected     77 

Glisson's  capsule     78 

Glycogen 126 

, ,         in  cells     79 

Glycosuria        172 

Grove  cell        185 

Guaiacum  test 137 

Hsemocytometer     148 

Hair,  follicle    85 

Haemoglobin,  crystals    62 

Hsemoglobinometer        151 

Hsemoglobinuria     171 

Hamiolymph  gland         87 

Hsemin  crystals       62 

Hayem's  fluid 61 

Heart,  apex  beat    240 

frog      245 

,,    atropine      248 

,,    crescent  (vagus)      247 
,,    heat  and  cold    ...  248 

,,    recorder      246 

, ,    latent  period      . . .  249 
Helmholtz  side  wire       192 


INDEX. 


271 


Induction,  current 

extra 
,,  ,,        make    and 

break     

Inductorium    

Interrupter  shocks  

,,  „       strength  of 

189, 
Intestine,  fat  absorption 

large        

,,         small       

,,       injected 

Irrigation 

Isometric  contraction    

Isotonic  .,  


Keys,  electrical       

Kjeldahl,  for  nitrogen  .. 
Kidney      

,,       injected       

,,       isolated  tubules 

Labelling  specimens 

Lactose     

Legal's  test      

Leelanche  cell 

Lens,  fibres      

Ligamentum  nucha? 

Linen  fibre       

Listing's  reduced  eye  . 
Liver  cells        

,,      ducts      

,,      glycogen        ...     . 
Lung,  feetal      

,,      injected 

,,      silvered 

Lymph  gland 

,,  injected  . 

Mammary  gland     ... 
Marrow     


AGE 

188 
191 

198 
188 
191 

201 
73 
74 
73 
74 
12 
219 
213 

185 
164 

80 
81 
82 


...  123 

...  128 

...  185 

...  103 

...  35 

...  12 

...  256 

...  78 

...  79 

...  79 

...  67 

...  67 

...  66 
36,  86 

...  86 

...  100 

...  43 


PAGE 

Measuring,  microscopical     ...       8 
Medulla  oblongata  (Bulb)     ...     'J.") 

Microscope,  student's    ~>.  <i 

,,  care  of        5,  6 

Microtome,  ether    25 

,,  horse  shoe 23 

,,  rocking        23 

Midbrain 96 

Milk 136 

,,    globules 13 

Millon's  test    130 

Mitosis      29 

Moore's  test    122 

Mucin        169 

Muscle,  cardiac       47 

chamber    205,  209 

,,        elasticity 212 

excitation 223,201 

,,        extensibility     212 

,,        fatigue       217 

,,        heat  and  cold 221 

,,        latent  period    213 

,,        load  on      218 

,,        non-striped       47 

,,        spindles     50 

,,        striped       48 

,,        tongue        63 

,,        nerve  preparation  ...   196 

Myograph 205,  209,  229 

Myosin      132 

Myosinogen      138 

Nail 85 

Nerve  cells      54 

change  in  excitability   203 

excitation    201 

medulla  ted 51 

osmic,  teased       51 

,,       section     53 


silvered 
non-medullated 


52 
53 


272 


INDEX. 


r.MiE 

PAGE 

Neuroglia 

...     97 

Rennet      

136 

Newt's  moult 

...     13 

Resistance,  electrical     ...  183 

,  263 

Nervous  impulse    

199,  226 

Respiratory  system        

65 

Retina       

103 

(Esophagus       

...     70 

,,      illuminated 

104 

Olfactory  epithelium     ... 

...  101 

,,      periodic  stimulation  ... 

257 

Opthalmoscope        

...  253 

Rheoscopic  limb     

199 

Optic  papilla 

...  103 

Rheocord 

233 

Ovary        

...     99 

Oxalate  of  lime      

...  169 

Saliva        

140 

Saponification 

127 

Pain  sense        

...  260 

Schemer's  experiment 

255 

Palate,  soft      

...     69 

Sections,  cutting,  celloidin  ... 

26 

Paradoxical  contraction 

...  200 

gum 

25 

Paraffin,  mounting  from 

14,  15 

,,              ,,          paraffin    ... 

20 

Parathyroid  body 

...     88 

,,         flotation  on  water  ... 

13 

Pendulum  mj^ograph     ... 

...  229 

Semi-circular  canals      

105 

PA  tiger's  law 

...  233 

Serum  proteids 

145 

Pettenkoffer's  test 

...  155 

Sinus  of  Valsalva  ... 

56 

Perimeter 

...  251 

Skin 

84 

Peyer's  patch 

...     74 

Slides,  to  clean 

7 

,,             ,,     injected  ... 

...     74 

,,      to  finish  off 

7 

Phenol,  hydrazin  test   ... 

...  122 

Spermatogenesis 



98 

Phosphoric  acid      

159,  137 

Spleen       

87 

vol 

...  161 

,,      injected 

88 

Pons  Varolii    

...     96 

Spinal  cord      

91 

Piotrowski's  test    

...  130 

,,           ,,    regional 



94 

Pituitary  body        

...     89 

Stannius'  ligature  ... 

249 

Polarisation      

...  183 

Starch  granules 

12 

Preserving  b}'  injection 

...     17 

, ,       soluble 

125 

Prostrate  

...     98 

,,       neutral  salts  on 

... 

125 

Proteids,  reactions 

...  129 

,,       digestion     ... 

..  125 

143 

,,         table  of  tests 

...  135 

Staining  on  the  slide 

13 

Pulse  tracing 

...  242 

,,         in  bulk    ... 
Stomach    

19 

70 

Quadriurates 

...  169 

Strassburg's  test    ... 
Sugar,  cane      

155 
123 

Reagents,  chemistry 

...     59 

Suprarenal  body     ... 

89 

Recording  arms      

...  206 

,,           effect  on  vessels 

250 

,,            cylinder 

...  205 

,,                 ,,       covering      206 

Temperature,  coagulati 

an    ... 

132 

index; 


273 


PAGE 

PAGB 

Tendon     

34 

Urine,  tesl  table1    ... 

177 

Testis       

98 

,,       salts      

158 

Tetanus     

216 

1  'Hilary  bladder 

83 

Thyroid     

ss 

Unipolar  stimulation 

203 

Time  distribution  l» 

>ard      ...  208 

Uterus       

LOO 

Tongue      

68 

Tonsil       

86 

Vagina     

100 

Tooth,  adult    

44 

Vas  deferens     

98 

„       growing 

45 

Ventricle,  structure 

57 

Touch       

259 

Veratria,  muscle    ... 

224 

Trachea    

65 

Vermiform  appendix 

74 

Tracings,  fixing 

210 

Urates      

168 

Wheatstone's  bridge 

263 

Urea 

161 

Whey        

136 

,,     estimation 

162 

Wool  fibre        

12 

Ureter       

82 

Work  table  histological 

...      7 

Uric  acid 

167 

,,          ,,     experimental     ...  204 

Urine         

157 

,,      abnormal 

170 

Xanthoproteic  reaction 

...  129 

CUORLBY   A    PlCKJERSGILL,  THE   ELECTRIC   PRESS,    LEEDS. 


